MMP-9 mRNA Expression Research Articles

Icariin-conditioned serum combined with chitosan attenuates cartilage injury in rabbit knees with osteochondral defect

BackgroundKnee osteoarthritis (KOA) is one of the most common degenerative diseases. Its development is closely related to cartilage injury and subchondral bone remodeling homeostasis. In the present study, we combined icariin-conditioned serum (ICS) with thiolated chitosan (CSSH), a material widely used in tissue engineering for cartilage repair, to demonstrate its effect on the repair of cartilage damage and abnormal subchondral remodeling.MethodsNew Zealand rabbits were undergoing surgery for cartilage defect, and joint cavity injection was performed in each group with 0.5 mL normal saline (NS), ICS, CSSH and ICS-CSSH in the right joint every week for five times. Positioning performance was observed using VICON motion capture system. Glycosaminoglycans (GAG) secretion of articular fluid was assessed. Osteoarthritis Research Society International (OARSI) score and immunohistochemical (IHC) analysis including H&E, Safranin O and collagen II staining were employed to evaluate the morphologic repair of cartilage and subchondral bone. mRNA expression of COL2A1, MMP13 and ADAMTS5 was detected in chondrocytes from injury area.ResultsICS combined with CSSH attenuated cartilage injury and abnormal subchondral remodeling in rabbits with KOA. ICS and CSSH groups showed slight improvement in positioning performance, while ICS-CSSH group exhibited better positioning performance. ICS-CSSH group showed increased GAG secretion of articular fluid and expression of COL2A1 in articular chondrocytes. Furthermore, both macroscopic observation and IHC analysis showed femoral condyle in ICS-CSSH rabbits were repaired with more native cartilage and subchondral bone regeneration.ConclusionsICS combined with CSSH could promote the repair of osteochondral defect and stabilize subchondral bone remodeling in rabbit knees.

Cornelian Cherry (Cornus mas L.) Iridoid and Anthocyanin-Rich Extract Reduces Various Oxidation, Inflammation, and Adhesion Markers in a Cholesterol-Rich Diet Rabbit Model.

Atherogenesis leads to the development of atherosclerosis, a progressive chronic disease characterized by subendothelial lipoprotein retention and endothelial impairment in the arterial wall. It develops mainly as a result of inflammation and also many other complex processes, which arise from, among others, oxidation and adhesion. Cornelian cherry (Cornus mas L.) fruits are abundant in iridoids and anthocyanins-compounds with potent antioxidant and anti-inflammatory activity. This study aimed to determine the effect of two different doses (10 mg and 50 mg per kg of body weight, respectively) of iridoid and anthocyanin-rich resin-purified Cornelian cherry extract on the markers that are important in the progress of inflammation, cell proliferation and adhesion, immune system cell infiltration, and atherosclerotic lesion development in a cholesterol-rich diet rabbit model. We used biobank blood and liver samples that were collected during the previous original experiment. We assessed the mRNA expression of MMP-1, MMP-9, IL-6, NOX, and VCAM-1 in the aorta, and the serum levels of VCAM-1, ICAM-1, CRP, PON-1, MCP-1, and PCT. The application of the Cornelian cherry extract at a dose of 50 mg/kg bw resulted in a significant reduction in MMP-1, IL-6, and NOX mRNA expression in the aorta and a decrease in VCAM-1, ICAM-1, PON-1, and PCT serum levels. The administration of a 10 mg/kg bw dose caused a significant decrease in serum ICAM-1, PON-1, and MCP-1. The results indicate the potential usefulness of the Cornelian cherry extract in the prevention or treatment of atherogenesis-related cardiovascular diseases, such as atherosclerosis or metabolic syndrome.

Investigating the chemical profile of Rheum lhasaense and its main ingredient of piceatannol-3′-O-β-D-glucopyranoside on ameliorating cognitive impairment

Rheum lhasaense A. J. Li et P. K. Hsiao, a stout herb plant from the Polygonaceae, is a typical Tibetan folk herb with heat-clearing and detoxifying effects, but does not have the typical laxative effect compared with other rhubarb plants. Nevertheless, its chemical composition and pharmacological activities still lack in-depth research. The present study endeavored to analyze the possible phytochemical constituents in R. lhasaense and explore the main compound piceatannol-3′-O-β-D-glucopyranoside (PG) effect on cognitive impairment and its underlying mechanism. The chemical profile of R. lhasaense discovered 46 compounds, including 27 stilbenoids and 13 gallotannins using UPLC-Q-TOF-MS/MS. The UPLC determined the contents of 6 main stilbenoids, among which the content of PG was the highest, up to 61.06 mg/g. Moreover, behavioral tests showed that PG (40 mg/kg and 160 mg/kg) administration markedly ameliorated memory impairments of scopolamine-induced mice. Biochemical parameters showed that PG treatment alleviated the levels of Ach, AchE, and inflammatory factors while elevating the levels of antioxidants in mice. In addition, network pharmacology was performed to reveal PG exert an mild cognitive impairment effect by participating in neurodegenerative disease pathways, proliferation and apoptosis-, and inflammation-related pathways. Eventually, the results of molecular docking and the qRT-PCR revealed that PG down-regulated the mRNA expressions of MMP3, MMP9 and BACE1 in cognitive impairment mice brain tissue. In conclusion, our results demonstrated that PG mitigated scopolamine-induced cognitive dysfunction in mice by targeting the BACE1-MMP3/9 pathway, and PG might be a promising mild AD drug candidate.

Luteolin Protects Against Obese Sarcopenia in Mice with High-Fat Diet-Induced Obesity by Ameliorating Inflammation and Protein Degradation in Muscles.

Although sarcopenia is mainly caused by aging, sarcopenia due to obesity has become an emerging issue given the increase in obesity among people of various ages. There are studies on obesity or sarcopenia, our understanding of obesity-mediated sarcopenia is insufficient. Luteolin (LU) has exhibited antiobesity effects, but no studies have investigated the LU effects on antisarcopenia. This study therefore investigated the effects of LU on obese sarcopenia in mice with high-fat diet (HFD)-induced obesity. To evaluate its inhibitory efficacy against obese sarcopenia, 5-week-old mice are fed an HFD supplemented with LU for 20weeks. LU exerts suppressive effects on obesity, inflammation, and protein degradation in the HFD-fed obese mice. It also inhibits lipid infiltration into the muscle and decreases p38 activity and the mRNA expression of inflammatory factors, including TNF-α, Tlr2, Tlr4, MCP1, and MMP2, in the muscle. The suppression of muscle inflammation by LU leads to the inhibition of myostatin, FoxO, atrogin, and MuRF expression. These effects of LU affect inhibition of protein degradation and improvement of muscle function. Here, it demonstrates that LU's antiobesity and antiinflammatory functionality affect inhibition of muscle protein degradation, and consequently, these interactions by LU exerts a protective effect against obese sarcopenia.

Ginsenoside Rg1 as a promising adjuvant agent for enhancing the anti-cancer functions of granulocytes inhibited by noradrenaline.

In recent years, numerous studies have confirmed that chronic stress is closely related to the development of cancer. Our previous research showed that high levels of stress hormones secreted in the body during chronic stress could inhibit the cancer-killing activity of granulocytes, which could further promote the development of cancer. Therefore, reversing the immunosuppressive effect of stress hormones on granulocytes is an urgent problem in clinical cancer treatment. Here, we selected noradrenaline (NA) as a representative stress hormone. After screening many traditional Chinese herbal medicine active ingredients, a promising compound, ginsenoside Rg1, attracted our attention. We verified the immunoprotective effect of ginsenoside Rg1 on granulocytes in vitro and ex vivo, and attempted to understand its potential immunoprotective mechanism. We confirmed the immunoprotective effect of ginsenoside Rg1 on granulocytes using cell and animal experiments. Cell counting kit-8 (CCK-8) and ex vivo experiments were performed to investigate the immunoprotective effects of ginsenoside Rg1 on the anti-cancer function of granulocytes inhibited by NA. Transcriptome sequencing analysis and qRT-PCR showed that NA elevated the mRNA expression of ARG2, MMP1, S100A4, and RAPSN in granulocytes, thereby reducing the anti-cancer function of granulocytes. In contrast, ginsenoside Rg1 downregulated the mRNA expression of ARG2, MMP1, S100A4, and RAPSN, and upregulated the mRNA expression of LAMC2, DSC2, KRT6A, and FOSB, thereby enhancing the anti-cancer function of granulocytes inhibited by NA. Transwell cell migration experiments were performed to verify that ginsenoside Rg1 significantly enhanced the migration capability of granulocytes inhibited by NA. Tumor-bearing model mice were used to verify the significant immunoprotective effects in vivo. Finally, CCK-8 and hematoxylin and eosin staining experiments indicated that ginsenoside Rg1 exhibited high biosafety in vitro and in vivo. In future clinical treatments, ginsenoside Rg1 may be used as an adjuvant agent for cancer treatment to alleviate chronic stress-induced adverse events in cancer patients.

Inhibitory effect of non-alcoholic steatohepatitis on colon cancer liver metastasis.

The incidence of non-alcoholic steatohepatitis (NASH) is dramatically increasing, but the effect of NASH on colon cancer liver metastasis (CLM) is controversial. The aim of this study was to investigate the impact and mechanism of action of NASH on CLM using a western diet (WD)-fed mouse model. Six-week-old male C57BL/6J mice were used. They were divided into the WD group and control group with normal diet. MC38 colon cancer cells were injected into the spleen at 2, 6, 8 and 16 weeks, and mice were killed at 2 weeks after injection to evaluate hepatic steatosis, fibrosis, metastasis and mRNA/protein expression in the liver. Only mice fed a WD for 16 weeks showed hepatic fibrosis. These mice showed significantly higher alanine aminotransferase and total cholesterol levels compared with the control group (p<0.05). The WD group showed significantly lower tumor number and smaller tumor diameter (p<0.05). In the WD group, expression of SAA1, IL6, STAT3 and MMP9 mRNA in the liver was significantly lower than in the control group (p<0.05). Serum amyloid A1 protein expression was also lower in the WD group. The WD-fed NASH mouse model showed an inhibitory effect on CLM. Suppressed interleukin-6/signal transducer and activator of transcription 3 signaling and serum amyloid A/matrix metalloproteinase 9 expression may affect this phenomenon.

Downregulated ETV4 inhibits the proliferation, migration, and invasion of trophoblast cells in preeclampsia.

Preeclampsia is a pregnancy complication that can lead to severe adverse maternal and fetal outcomes, but the mechanisms underlying the development of preeclampsia are not fully understood. This study shows that ETV4 plays an essential role in the proliferation, invasion, and migration of trophoblast cells by regulating MMP-2 and MMP-9 and is involved in the pathogenesis of preeclampsia. Preeclampsia (PE) is a pregnancy complication that can lead to severe adverse maternal and fetal outcomes. However, the mechanisms underlying the development of PE are not fully understood. ETS Variant Transcription Factor 4 (ETV4) plays an important role in cell proliferation, migration, and invasion. In this study, we aimed to explore the potential function of ETV4 in placental trophoblast cells. We analyzed the expression and location of ETV4 in PE and uncomplicated placental tissues using RT-qPCR, Western blotting, immunohistochemistry, and immunofluorescence staining. The results showed that both the mRNA and protein levels of ETV4 were markedly decreased in PE placental tissues compared with placental tissues from women with uncomplicated pregnancies (P < 0.05). Then, the effects of ETV4 on HTR-8/SVneo and Bewo cell proliferation, migration, and invasion were evaluated by MTT, 5-ethynyl-2-deoxyuridine (EdU), wound healing, and Transwell assays, respectively. The results showed that ETV4 knockdown inhibited both HTR-8/SVneo and Bewo cell proliferation, migration, and invasion (P < 0.05). Conversely, overexpression of ETV4 promoted both HTR-8/SVneo and Bewo cell proliferation, migration, and invasion (P < 0.05). We then measured the expression of MMP-2 and MMP-9 in HTR8/SVneo cells. We found that ETV4 knockdown decreased the mRNA and protein expression of MMP-2 and MMP-9, while ETV4 overexpression increased MMP-2 and MMP-9 mRNA and protein expression (P < 0.05). In conclusion, ETV4 plays an essential role in the proliferation, invasion, and migration of trophoblast cells by regulating MMP-2 and MMP-9. Our findings provide novel insight into the mechanisms underlying the occurrence of PE.

P012 The administration of mm-miR-378a-3p exacerbates chronic inflammation and fibrosis in a murine intestinal model

Abstract Background Fibrosis constitute an important complication of CD. MicroRNAs (miRNAs), which are small RNA molecules that regulate gene expression, have been shown to participate in the molecular interactions of both inflammation and fibrosis. Lower levels of miR-378a-3p have been reported associated to murine liver fibrosis (Hyun et al. DOI: 10.1038/ncomms10993) and we analyze here the relevance of miR-378a-3p on intestinal fibrosis. Methods B57BL/6 mice were intravenously injected with 2,5mg/kg of negative control (NC) or mm-miR-378a-3p mimic, twice a week and received vehicle or Dextran Sulfate Sodium (DSS) for 2 cycles (7 days drinking DSS 2% in water solution followed by 10 days drinking water). Body weight and DAI score was obtained every day and the colon was collected after sacrifice. Sirius and hematoxylin-eosin dyes were employed to determine the fibrosis and structural state in 5µm slides of intestinal tissue. Gene expression and miRNA profiles were analyzed by RT-qPCR. Human small intestinal fibroblasts (HSIF; P10760, Innoprot, Spain) were transfected with 20nM of NC or hsa-miR-378a-3p mimic during 24h. Results No significant changes in body weight, DAI score, and colon length were detected between mice receiving NC and those receiving mimic, all along the two DSS cycles. Colon of mice treated with DSS, exhibited a significant diminution in the mRNA expression of mm-miR-378a-3p compared with naïve samples. In DSS-treated mice, the iv administration of the mimic compared with the NC: a) significantly increased levels of miR-378a-3p in the colon; b) increased the number of neutrophils, and heightened changes in the glandular epithelia and architectural distortion (figure 1a) C) decreased the number of lymphoid follicles (figure 1a); d) slightly increased collagen deposition, as analyzed by sirius red (figure 1b) and e) significantly increased the mRNA expression of Tgfb1, Il1b, and Mmp2. Treatment of human intestinal fibroblasts with hsa-miR-378a-3p mimic did not significant modify the mRNA expression of markers of fibrosis but it significantly increased the mRNA expression of two antiapoptotic molecules BCL2 and MCL1. Conclusion Levels of mm-miR-378a-3p are diminished in a murine model of intestinal fibrosis; The exogenous administration of miR-378a-3p, increased the acute inflammatory response and the expression of fibrosis markers in murine fibrotic colon and the expression of antiapoptotic molecules in human intestinal fibroblasts.

Wumei Wan attenuates angiogenesis and inflammation by modulating RAGE signaling pathway in IBD: Network pharmacology analysis and experimental evidence

BackgroundWumei Wan (WMW) has been used to address digestive disorder for centuries in traditional Chinese medicine. Previous studies have demonstrated its anti-colitis efficacy, but the underlying mechanism of its action remains to be further clarified. PurposeTo investigate the underlying mechanisms of WMW in the treatment of chronic ulcerative colitis (UC) through network pharmacology and experimental validation. MethodsTraditional Chinese Medicine Systems Pharmacology (TCMSP) platform were used to identify the ingredients and potential targets of WMW. The microarray gene data GSE75214 datasets from GEO database was used to define UC-associated targets. Cytoscape3.7.2 was employed to construct the protein-protein interaction (PPI) network and compounds-disease targets network. GO enrichment analysis and KEGG pathway analysis were performed by R software for functional annotation. UPLC-TOF-MS/MS method was used to quantitatively analyze the active ingredients of WMW. For experimental validation, three cycles of 2% dextran sulfate sodium salt (DSS) were used to construct chronic colitis model. The hub targets and signal pathway were detected by qPCR, ELISA, western blotting , immunohistochemical and immunofluorescence. ResultsThrough network analysis, 104 active ingredients were obtained from WMW, and 47 of these ingredients had potential targets for UC. A total of 41 potential targets of WMW and 13 hub targets were identified. KEGG analysis showed that WMW involved in advanced glycation end products-receptor of advanced glycation end products (AGE-RAGE) signaling pathway. Taxifolin, rutaecarpine, kaempferol, quercetin, and luteolin of WMW were the more highly predictive components related to the AGE-RAGE signaling pathway. In vivo validation, WMW improved DSS-induced colitis, reduced the expression of inflammatory cytokines and chemokines. Notably, it significantly decreased the mRNA expression of Spp1, Serpine1, Mmp2, Mmp9, Ptgs2, Nos2, Kdr and Icam1, which were associated with angiogenesis. In addition, we confirmed WMW inhibited RAGE expression and diminished DSS-induced epithelial barrier alterations ConclusionOur results initially demonstrated the effective components and the strong anti-angiogenic activity of WMW in experimental chronic colitis. Sufficient evidence of the satisfactory anti-colitis action of WMW was verified in this study, suggesting its potential as a quite prospective agent for the therapy of UC.

Lisinopril prevents bullous pemphigoid induced by dipeptidyl peptidase 4 inhibitors via the Mas receptor pathway.

Recent studies have suggested that dipeptidyl peptidase 4 (DPP4) inhibitors increase the risk of development of bullous pemphigoid (BP), which is the most common autoimmune blistering skin disease; however, the associated mechanisms remain unclear, and thus far, no therapeutic targets responsible for drug-induced BP have been identified. Therefore, we used clinical data mining to identify candidate drugs that can suppress DPP4 inhibitor-associated BP, and we experimentally examined the underlying molecular mechanisms using human peripheral blood mononuclear cells (hPBMCs). A search of the US Food and Drug Administration Adverse Event Reporting System and the IBM® MarketScan® Research databases indicated that DPP4 inhibitors increased the risk of BP, and that the concomitant use of lisinopril, an angiotensin-converting enzyme inhibitor, significantly decreased the incidence of BP in patients receiving DPP4 inhibitors. Additionally, in vitro experiments with hPBMCs showed that DPP4 inhibitors upregulated mRNA expression of MMP9 and ACE2, which are responsible for the pathophysiology of BP in monocytes/macrophages. Furthermore, lisinopril and Mas receptor (MasR) inhibitors suppressed DPP4 inhibitor-induced upregulation of MMP9. These findings suggest that the modulation of the renin-angiotensin system, especially the angiotensin1-7/MasR axis, is a therapeutic target in DPP4 inhibitor-associated BP.

Development on potential skin anti-aging agents of Cosmos caudatus Kunth via inhibition of collagenase, MMP-1 and MMP-3 activities

BackgroundSkin aging is associated with degradation of collagen by matrix metalloproteinases (MMPs), which leads to loss of skin elasticity and formation of wrinkles. Cosmos caudatus Kunth (CC) has been traditionally claimed as an anti-aging agent in Malaysia. Despite its well-known antioxidant activity, the anti-aging properties of CC was not validated. PurposeThis study aimed to investigate the anti-aging potential of CC extracts and fractions, particularly their inhibition of collagenase, MMP-1 and MMP-3 activities in human dermal fibroblasts CCD-966SK, followed by isolation, identification and analysis of their bioactive constituents. Study design and methodsDPPH assay was firstly used to evaluate the antioxidant activity throughout the bioactivity-guided fractionation. Cell viability was determined using MTS assay. Collagenase activity was examined, while MMP-1 and MMP-3 expression were measured using qRT-PCR and western blotting. Then, chemical identification of pure compounds isolated from CC fractions was done by using ESIMS, 1H and 13C NMR spectroscopies. HPLC analyses were carried out for bioactive fractions to quantify the major components. ResultsThroughout the antioxidant activity-guided fractionation, fractions CC-E2 and CC-E3 with antioxidant activity and no toxicity towards CCD-966SK cells were obtained from CC 75% ethanol partitioned layer (CC-E). Both fractions inhibited collagenase activity, MMP-1 and MMP-3 mRNA and protein expression, as well as NF-κB activation induced by TNF-α in CCD-966SK cells. 14 compounds, which mainly consists of flavonoids and their glycosides, were isolated. Quercitrin (14.79% w/w) and quercetin (11.20% w/w) were major compounds in CC-E2 and CC-E3, respectively, as quantified by HPLC. Interestingly, both fractions also inhibited the MMP-3 protein expression synergistically, compared with treatment alone. ConclusionThe quantified CC fractions rich in flavonoid glycosides exhibited skin anti-aging effects via the inhibition of collagenase, MMP-1 and MMP-3 activities, probably through NF-κB pathway. This is the first study reported on MMP-1 and MMP-3 inhibitory activity of CC with its chemical profile, which revealed its potential to be developed as anti-aging products in the future.

Antiangiogenic Potential of Troxerutin and Chitosan Loaded Troxerutin on Chorioallantoic Membrane Model.

Angiogenesis is crucial to the development of cancer because it allows the transport of oxygen, nutrients, and growth factors as well as the spread of tumors to distant organs. Inhibitors of angiogenesis prevent the formation of blood vessels that allow tumor cells to shrink, rather than promote tumor growth. Chitosan acts as a carrier for many drugs, since the compound has various properties such as biodegradable, less toxicity, more stable, simple, easy to prepare, and biocompatible. The aim of the current study was to evaluate the efficacy of chitosan nanoparticles encapsulated with troxerutin (Chi-Trox NPs) against angiogenesis and cancer in ova chick chorioallantoic membrane (CAM) model. Chi-Trox NPs were synthesized using a nanoprecipitation method and were characterized by various analyses. 24 hours' fertilized eggs (6 eggs/group) were treated with native Trox and Chi-Trox NPs for 5 days. The antiangiogenic activity was evaluated by morphometric, histopathological, immunohistochemical (CD104 and vimentin), and mRNA expression of MMP and FGF2 using RT-PCR. The anticancer activity was evaluated by histopathological, immunohistochmical (CD44), and mRNA expression of FGF2 and MMP. The synthesized chitosan NPs were successfully encapsulated with troxerutin, and the loading efficiency of chitosan NPs was found to be 86.4 ± 0.12% and 13.2 ± 0.16% respectively. Morphometric analysis of Chi-Trox NPs showed a considerable decrease in the number of blood vessels compared with control and native Trox. The histopathological observation of CAM confirmed that Chi-Trox NPs induce a significant reduction in inflammatory cells and the thickness of blood capillaries compared to control and native Trox. The immunohistochemical evaluation of CAM revealed Chi-Trox decreased CD104, vimentin and CD44 protein levels were compared with control and native Trox. Furthermore, the mRNA expression levels of FGF2 and MMP were significantly downregulated compared to their native forms. From the obtained results, Chi-Trox NPs possess significant inhibition of angiogenesis and can be used as therapeutic agents for cancer in the future.

Wogonin Restrains the Malignant Progression of Lung Cancer Through Modulating MMP1 and PI3K/AKT Signaling Pathway.

Wogonin, a natural flavonoid compound, represses cancer cell growth and induces cancer cell apoptosis in diverse malignancies. However, the function of Wogonin in lung cancer cells and its regulatory mechanism deserve to be identified. A549 and H460 cells were treated with Wogonin, and the cell growth, apoptosis, migration and invasion were measured by CCK-8 and EdU, flow cytometry and Transwell assays. The targeted genes of Wogonin and lung cancer were identified from the TCMSP and Genecards databases, respectively. The STRING database and Cytoscape software were used to establish a PPI network and screen hub genes. GO and KEGG analysis was conducted to explore the functions and signal pathways related to the hub genes. MMP1 expression in lung cancer was analyzed using the UALCAN databases, and GSEA was performed utilizing LinkedOmics. Gelatin zymography assay was used to detect MMP1 activity. MMP1 mRNA expression was detected by qRT-PCR. Besides, MMP1, p-AKT and c-Myc protein were detected by Western Blot assay. Wogonin could suppress the proliferation, migration and invasion of A549 and H460 cells and induce apoptosis. GO and KEGG enrichment analysis revealed the hub genes were mostly enriched in re-entry into the mitotic cell cycle and apoptosis. The expression of MMP1 was markedly upregulated in lung squamous cell carcinoma, lung adenocarcinoma tissues, and lung cancer cell lines. Wogonin could significantly inhibit MMP1 expression and activity, and overexpression of MMP1 significantly reversed the effect of Wogonin on the malignant phenotypes of A549 and H460 cells. Wogonin inhibited the expression of p-AKT and c-Myc protein by regulating MMP1. Wogonin can repress lung cancer cells' growth and metastatic potential and promote cell apoptosis via repressing MMP1 expression and modulating PI3K/AKT signaling pathway.

Quercetin inhibits body weight gain and adipogenesis via matrix metalloproteinases in mice fed a high-fat diet.

Limited studies reported that quercetin inhibited adipogenesis and neovascularization by inhibiting matrix metalloproteinases (MMPs) activity, but such mechanisms have not been elucidated in animal experiments. In this study, we investigated the inhibitory effects of quercetin on weight gain and adipose tissue growth through the regulation of mRNA expressions of adipogenic transcription factors and MMPs in mice fed a high-fat diet (HFD). Five-wk-old C57BL/6J mice were fed a normal diet (ND), HFD, HFD containing 0.05% of quercetin (HFQ0.05), or HFD containing 0.15% of quercetin (HFQ0.15) for 16 wks. Glycerol-3-phosphate dehydrogenase (GPDH) activity was measured using a commercial kit. The mRNA expressions of transcription factors related to adipocyte differentiation were determined by real-time polymerase chain reaction (PCR). The mRNA expressions of MMPs and concentrations of MMPs were measured by real-time PCR and enzyme-linked immunosorbent assay kit, respectively. Quercetin intake reduced body weight gain and epididymal adipose tissue weights (P < 0.05). GPDH activity was higher in the HFD group than in the ND group but lower in the quercetin groups (P < 0.05). The mRNA expressions of CCAAT/enhancer binding protein β (C/EBPβ), C/EBPα, peroxisome proliferator-activated receptor γ, and fatty acid-binding protein 4 were lower in the quercetin groups than in the HFD group (P < 0.05). Similarly, the mRNA expression and concentrations of MMP-2 and MMP-9 were significantly lower in the quercetin groups than in the HFD group (P < 0.05). The study confirms that quercetin suppresses body weight gain and adipogenesis by inhibiting transcription factors related to adipocyte differentiation and MMPs (MMP-2 and MMP-9), in mice fed a HFD.

Resveratrol suppresses lipopolysaccharide-mediated activation of osteoclast precursor RAW 264.7 cells by increasing miR-181a-5p expression

Resveratrol (Res) has anti-inflammation and antiosteoporosis functions. We evaluated the effect of Res on osteoclast differentiation by releasing inflammatory cytokines from osteoclast precursor RAW 264.7 cells stimulated by lipopolysaccharide (LPS). In the study, LPS (1ng/L) was used to induce the Raw 264.7 inflammatory injury model in vitro. A total of 25ng/mL M-CSF + 30ng/mL RANKL or plus 1μg/L LPS was used to induce osteoclastogenesis in the experiments. We utilized the Cell Counting Kit-8 assay to measure the relative cell survival of RAW 264.7 cells. Then, enzyme-linked immunosorbent assays were utilized to measure the abundance of inflammatory markers, such as interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and IL-6. Subsequently, Western blot analysis was applied to assess the abundance of phosphorylated transforming growth factor beta-activated kinase 1 (P-TAK1) protein, TNF receptor-associated factor 6 (TRAF6), nuclear factor-κB inhibitor protein (IκB), phosphorylated IκB-α(P-IκB-α), and nuclear factorκB65 (NF-κB65). mRNA expression levels of miR-181a-5p, TRAF6, specific gene calcitonin receptor (CTR), activated T nuclear factor 1 (NFATC1), cathepsin K (CTSK), and matrix metalloproteinase (MMP)-9 were determined via a real-time polymerase chain reaction. Osteoclast bone resorption function was determined. Finally, tartrate-resistant acid phosphatase (TRAP) staining was performed.The results found that Compared with the model group, the degrees of expressions of supernatant inflammatory factors TNF-α, IL-1β, and IL-6 were substantially attenuated in the Res treatment group (p< 0.05). Furthermore, the extent of miR-181a-5p expression in the RAW 264.7 cells significantly increased, whereas P-IκB-α, P-TAK1, NF-κB65, and TRAF6 expressions significantly decreased in the Res treatment group as opposed to the model group (p< 0.05). The CTR, NFATC1, MMP-9, CTSK, and TRAP mRNA expression levels were substantially reduced during osteoclast differentiation and bone resorption in the Res treatment group.The results suggest that Res can reduce the RAW 264.7 cell differentiation into osteoclasts and relieve LPS-stimulated osteoporosis, and the underlying mechanism may be associated with the Res-inhibited activity of the TRAF6/TAK1 pathway through the increased miR-181a-5p expression.

The effect of natural products combination on MCF-7 cells exceeds tamoxifen therapeutic dose effects in vitro

Cancer remains to be one of the most severe sicknesses globally. Cases have kept rising over the years. Breast cancer (BC), which is among the leading types of cancers and predominantly affects women, is the second leading cause of cancer mortality. Researchers have developed interventions over the years; however, the BC survival rate has not improved since the 1980s. This has created the need for novel drug interventions that would manage and treat BC more effectively. This study focused on using a combination of natural product extracts such as phytoestrogen (Ziziphus jujube) and Tannin nanoparticles (NP99) together, which we have referred to as (Z.NP99) and tamoxifen (Tam) as one of the leading BC drugs since the 70s, in treating BC. The effectiveness of Tam if used alone in the treatment and if combined with Z.NP99 was evaluated using MCF-7 cells in vitro. The findings showed that the combination treatment of Z.NP99 affected the proliferation and viability of MCF-7 cells more than Tam at a 10 μg/mL dose. Moreover, Z.NP99 with Tam stimulated the maximum reduction of MCF-7 proliferation and viability in a time-dependent manner. Furthermore, Tam and Z.NP99 augmented the DNA fragmentation percentage combined with the upregulation of the apoptotic genes. Additionally, the results showed that the apoptotic impact of Z.NP99 and Tam on MCF-7 cells may be intermediated by down-regulating some genes such as Claudin-1 followed by down-regulating mRNA expression of MMP-9, VEGF, and BCL-2 genes of treated cells. Combining Tam with Z.NP99 considerably enhanced the effectiveness of conventional therapy. As a result, this study suggested that the Z.NP99 was ideal for developing effective natural treatments that would improve BC outcomes.

PS-BPB01-3: ANGIOTENSIN (1–7)-MEDIATED PREVENTIVE EFFECT OF ANGIOTENSIN II TYPE 1 RECEPTOR ANTAGONISTS ON ABDOMINAL AORTIC ANEURYSM PROGRESSION IN OPG-KO MICE

Objective: Hypertension is one of the major risk factors for onset of abdominal aortic aneurysm (AAA), which is a local enlargement of the abdominal aorta. Therefore, anti-hypertensive drugs, including angiotensin (Ang) II type 1 receptor blocker (ARB), have been expected to limit AAA growth. However, the efficacy of ARB against AAA in clinical settings is controversial. In the present study, we attempted to elucidate a mechanism by which ARB inhibits AAA progression. Design and method: To examine the effect of ARB against AAA progression, olmesartan (20 mg/kg/day) was administered orally to male wild-type mice and osteoprotegerin (Opg)-knockout (KO) mice, from two weeks before the AAA induction by using a CaCl2 application on the abdominal aortae to the sacrifice date. Since Opg could bind to tumor necrosis factor-related apoptosis-inducing ligand (Trail) for inhibiting its activity, deficiency of the Opg gene has been reported to induce overactivation of the Trail induced-c-Jun N-terminal kinase (Jnk)/matrix metalloproteinase 9 (Mmp9) pathway in CaCl2-induced mouse AAA model, resulting in serious AAA progression. Here, we tested whether olmesartan could prevent AAA progression associated with Trail/Jnk/Mmp9 pathway. Results: We found that olmesartan prevented excessive aortic enlargement and serious degenerative change of the aortic tissue in Opg-KO mice at six weeks after AAA induction, but not affected AAA formation in wild-type mice. In Opg-KO mice, olmesartan reduced co-localized expression of phosphorylated Jnk and Mmp9 in the tunica media. Then, we tested whether Ang (1–7), the blood level of which is increased by ARB administration, is associated with the preventive effect of olmesartan, because Ang (1–7) had been reported to reduce the Jnk phosphorylation. Consistent with previous studies, olmesartan increased serum level of Ang (1–7) in our AAA model. In vascular smooth muscle cells, Ang (1–7) inhibited the phosphorylation of Jnk and the increase of downstream Mmp9 mRNA expression induced by Trail. Furthermore, administration of an Ang (1–7) antagonist, A779, to Opg-KO mice diminished the preventive effect of ARB against the AAA progression. Notably, olmesartan slightly lowered blood pressure in Opg-KO mice, while A779 did not affect the blood pressure-lowering effect of olmesartan. Conclusions: Our results indicate that ARB prevents the AAA progression in Opg-KO mice via the inhibition of Trail-induced Jnk-Mmp9 pathway. Furthermore, it is suggested that the increase of Ang (1–7) may play a key role in the preventive effect of ARB on the AAA progression.

Simvastatin-loaded PCL/PEG nanofibrous scaffold: A prospective approach for suppression 5-fluorouracil resistance in MKN-45 gastric cancer cells

BackgroundBecause the development of chemoresistance is a key issue in the treatment of gastric cancer (GC), there has been significant interest in combination chemotherapy. In this study, we examined whether simvastatin-loaded Polycaprolactone– Polyethylene Glycol nanofibers (SIM-PCL/PEG NFs) inhibit 5-fluorouracil (5-FU) resistance in MKN-45 gastric cancer cells. Methods5-FU-resistant MKN-45 cells (MKN-45/R) were established by increasing concentrations of SIM. SIM-PCL/PEG NFs were synthesized and characterized through SEM and FTIR testing. SIM-PCL/PEG NFs cytotoxicity on MKN-45 and MKN-45/R cells was determined using the MTT assay. mRNA expression of P53, Bax, Bcl-2, ULK1, Beclin1, LC3, MMP2, and MMP9 genes in both cell groups was measured by RT-PCR. Flow cytometry, DAPI staining, and colony formation assay were used to assess the anti-proliferative activity of SIM-PCL/PEG NFs on cells. Autophagy-related proteins and cell migration were evaluated using western blotting and wound healing assay, respectively. The STRING database was used for bioinformatics analyses. ResultsMTT assay showed that SIM-PCL/PEG NFs had a more cytotoxic effect on MKN-45/R cells. Flow cytometry analysis indicated that SIM-PCL/PEG NFs could cause more cell apoptosis in MKN-45/R cells and also, the mRNA expression levels showed significantly elevated p53 and Bax levels and reduced Bcl-2 in MKN-45/R cells in this group. Analysis of ULK1, Beclin1, and LC3 at the transcription and translation levels indicated SIM-loaded NFs promote autophagy in MKN-45/R cells. Furthermore, compared with the control group colony formation and migration of MKN-45/R cells in the SIM-loaded NFs group were significantly inhibited (all P < 0.05). ConclusionWe have introduced simvastatin-loaded nanofibres as a promising therapeutic compound for the inhibition of 5-FU resistance in gastric cancer cells.

Sidaxue, a traditional Guizhou Miao herbal medicine formula, reduces necroptosis and synovial vascular proliferation in rats with collagen-induced arthritis by inhibiting the RIPK1/RIPK3/MLKL pathway

To explore the inhibitory effect of Sidaxue (SX), a traditional Guizhou Miao herbal medicine formula, on necrotic apoptosis and synovial angiogenesis in rats with collagen-induced arthritis (CIA) and the role of the RIPK1/RIPK3/MLKL pathway in mediating this effect. Forty-two SD rats were randomized into 6 groups (n=7), including a normal control group, a CIA model group, 3 SX treatment groups at low (10 g/kg), moderate (20 g/kg) and high (40 g/kg) doses, and a GTW treatment group. CIA rat models were established by subcutaneous injections of bovine type II collagen, and the treatments were administered daily by gavage for 21 days. The rats were observed for swelling of the hind limb joints, which was rated using the arthritis index (AI) score on a weekly basis. Serum levels of TNF-α, IL-1β and IL-17 in the rats were detected using ELISA, and the pathological changes in the synovium were observed with HE staining. Real-time PCR was performed to detect the mRNA expression levels of VEGF, MMP-9, Ang-1, RIPK1, RIPK3, and caspase-8 in the synovial tissues, and the protein expressions of VEGF, MMP9, Ang-1, Stat-3, RIPK1, RIPK3, MLKLl, p-MLKL and caspase-8 were detected using Western blotting. Compared with those in CIA model group, the rats receiving treatment with GTW and SX showed milder swelling of the hind limb joints with significantly lower AI scores (P < 0.05). In CIA model group, a large number of inflammatory cells were observed in the synovium with obvious damages of the tissue structure. In the drug treatment groups, inflammatory cell infiltration, synovial angiogenesis and synovial hyperplasia were alleviated, and the therapeutic effects were obviously enhanced as SX dose increased. Compared with those in the model group, the rats treated with GTW and high-dose SX showed significantly decreased serum levels of IL-1β, IL-17 and TNF-α (P < 0.05), lower mRNA and protein expressions of RIPK1, RIPK3, VEGF, Ang-1, and MMP9 (P < 0.05), higher expressions of caspase-8 (P < 0.01), and obviously lowered expression of Stat-3 protein and phosphorylation level of MLKL (P < 0.05). SX can improve synovial angiogenesis in CIA rats possibly by inhibiting the activation of RIP1/RIP3/MLKL signaling pathway and down-regulating the expression of the vascular growth factors VEGF, Ang-1, MMP9, and Stat-3.

Effect of Secretome-Hypoxia Mesenchymal Stem Cells on Regulating SOD and MMP-1 mRNA Expressions in Skin Hyperpigmentation Rats

BACKGROUND: Ultraviolet B (UVB) radiation is the main factor causing hyperpigmentation. MSC secretome contains bioactive soluble molecules such as cytokines and growth factors that can accelerate skin regeneration. However, the molecular role of the secretome in hyperpigmentation is still unclear. AIM: This study aimed to determine the effect of secretome hypoxia mesenchymal stem cells (S-HMSC) gel on the expression of superoxide dismutase (SOD) and matrix metalloproteinases (MMP-1) genes in skin tissue of hyperpigmented rats induced by UVB light exposure. MATERIALS AND METHODS: Experimental research with post-test only control group. The control, base gel, T1 and T2 groups were UVB irradiated 6 times in 14 days at 302 nm with an minimal erythema dose of 390 mJ/cm2, respectively, while sham group did not receive UVB exposure. T1 was given 100 uL of S-HMSC gel and T2 was given 200 uL of S-HMSC gel every day for 14 days, while base gel received base gel. On day 15, skin tissue was isolated and analyzed for SOD and MMP-1 expression using qRT-PCR. RESULTS: The relative expression of the SOD gene in the treatment group (P1 = 0.47 ± 0.20, P2 = 1.22 ± 0.47) increased with increasing dose compared to the control group (UVB = 0.05 ± 0.01, Base gel = 0.05 ± 0.02). The relative expression of the MMP-1 gene in the treatment group (T1 = 5.82 ± 1.16, T2 = 2.86 ± 1.57) decreased with increasing dose compared to the control group (Control = 10.10 ± 2.31, and Base gel = 9.55 ± 1.29). CONCLUSION: Administration of S-HMSC gel can increase SOD gene expression and decrease MMP-1 gene expression in skin tissue of hyperpigmented rats model induced by UVB light.