Gelatin Microspheres Based on H8-Loaded Macrophage Membrane Vesicles to Promote Wound Healing in Diabetic Mice.

Diabetic wound healing remains a worldwide challenge for both clinicians and researchers. The high expression of matrix metalloproteinase 9 (MMP9) and a high inflammatory response are indicative of poor diabetic wound healing. H8, a curcumin analogue, is able to treat diabetes and is anti-inflammatory, and our pretest showed that it has the potential to treat diabetic wound healing. However, H8 is highly expressed in organs such as the liver and kidney, resulting in its unfocused use in diabetic wound targeting. (These data were not published, see Table S1 in the Supporting Information.) Accordingly, it is important to pursue effective carrier vehicles to facilitate the therapeutic uses of H8. The use of H8 delivered by macrophage membrane-derived nanovesicles provides a potential strategy for repairing diabetic wounds with improved drug efficacy and fast healing. In this study, we fabricated an injectable gelatin microsphere (GM) with sustained MMP9-responsive H8 macrophage membrane-derived nanovesicles (H8NVs) with a targeted release to promote angiogenesis that also reduces oxidative stress damage and inflammation, promoting diabetic wound healing. Gelatin microspheres loaded with H8NV (GMH8NV) stimulated by MMP9 can significantly facilitate the migration of NIH-3T3 cells and facilitate the development of tubular structures by HUVEC in vitro. In addition, our results demonstrated that GMH8NV stimulated by MMP9 protected cells from oxidative damage and polarized macrophages to the M2 phenotype, leading to an inflammation inhibition. By stimulating angiogenesis and collagen deposition, inhibiting inflammation, and reducing MMP9 expression, GMH8NV accelerated wound healing. This study showed that GMH8NVs were targeted to release H8NV after MMP9 stimulation, suggesting promising potential in achieving satisfactory healing in diabetic treatment.

Acetylated oligopeptide and N-acetylcysteine protect against iron overload-induced dentate gyrus hippocampal degeneration through upregulation of Nestin and Nrf2/HO-1 and downregulation of MMP-9/TIMP-1 and GFAP.

Iron accumulation in the brain causes oxidative stress, blood-brain barrier (BBB) breakdown, and neurodegeneration. We examined the preventive effects of acetylated oligopeptides (AOP) from whey protein on iron-induced hippocampal damage compared to N-acetyl cysteine (NAC). This 5-week study used 40 male albino rats. At the start, all rats received 150 mg/kg/day of oral NAC for a week. The 40 animals were then randomly divided into four groups: Group I (control) received a normal diet; Group II (iron overload) received 60 mg/kg/day intraperitoneal iron dextran 5 days a week for 4 weeks; Group III (NAC group) received 150 mg/kg/day NAC and iron dextran; and Group IV (AOP group) received 150 mg/kg/day AOP and iron dextran. Enzyme-linked immunosorbent assay, spectrophotometry, and qRT-PCR were used to measure MMP-9, tissue inhibitor metalloproteinase-1 (TIMP-1), MDA, reduced glutathione (GSH) levels, and nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) gene expression. Histopathological and immunohistochemical detection of nestin, claudin, caspase, and GFAP was also done. MMP-9, TIMP-1, MDA, caspase, and GFAP rose in the iron overload group, while GSH, Nrf2, HO-1, nestin, and claudin decreased. The NAC and AOP administrations improved iron overload-induced biochemical and histological alterations. We found that AOP and NAC can protect the brain hippocampus from iron overload, improve BBB disruption, and provide neuroprotection with mostly no significant difference from healthy controls.

Kechuanning Gel Plaster Exerts Anti-inflammatory and Immunomodulatory Effects on Ovalbumin-induced Asthma Model Rats via ERK Pathway.

BACKGROUND: We aimed to evaluate the therapeutic effects of Kechuanning gel plaster on ovalbumin (OVA)-induced rat model of asthma. METHODS: Rats were injected with OVA to induce asthma, and Kechuanning gel plaster was administered after the OVA challenge. The immune cell counts in the bronchial alveolar lavage fluid (BALF) were calculated after Kechuanning gel plaster administration. The levels of immune factors in BALF and serum OVA-specific IgE levels were analyzed. Western blot analysis and immunohistochemistry were carried out to analyze the following proteins: C-FOS, C-JUN, RAS p21 protein activator 1 (RASA1), matrix metalloproteinase 9 (MMP9), RAF1, p-MEK1, tissue inhibitor of metalloproteinase-1 (TIMP1), and p-extracellular signal-regulated kinase 1 (ERK1). RESULTS: Administration of Kechuanning gel plaster led to decreased immune cell counts, inflammatory cytokines (interleukin (IL)-1ß, IL13, and IL17), and OVA-specific IgE expression. Compared to the normal group, the C-FOS, C-JUN, RASA1, MMP9, RAF1, MEK1, TIMP1, and p- ERK1 expressions in the model group were significantly increased, whereas Kechuanning gel plaster administration decreased C-JUN, MMP9, TIMP1, RAF1, MEK1, p-ERK1, C-FOS, and RASA1 protein levels. CONCLUSION: Kechuanning gel plaster exerted its therapeutic effects on OVA-induced asthma model rats through the ERK signaling pathway. Kechuanning gel plaster could be considered as a potential alternative therapeutic agent for the management of asthma.

FXR agonists INT-787 and OCA increase RECK and inhibit liver steatosis and inflammation in diet-induced ob/ob mouse model of NASH.

BACKGROUND AND AIMS: We have previously shown in a model of hepatic ischaemia/reperfusion injury that the farnesoid X receptor (FXR) agonist obeticholic acid (OCA) restores reversion-inducing-cysteine-rich protein with Kazal motifs (RECK), an inverse modulator of metalloproteases (MMPs) and inhibitor of the sheddases ADAM10 and ADAM17 involved in inflammation and fibrogenesis. Here, the effects of FXR agonists OCA and INT-787 on hepatic levels of RECK, MMPs, ADAM10 and ADAM17 were compared in a diet-induced ob/ob mouse model of non-alcoholic steatohepatitis (NASH). METHODS: Lep ob/ob NASH mice fed a high-fat diet (HFD) or control diet (CD) for 9 weeks (wks) were treated with OCA or INT-787 0.05% dosed via HFD admixture (30 mg/kg/day) or HFD for further 12 wks. Serum alanine transaminase (ALT) and inflammatory cytokines, liver RECK, MMP-2 and MMP-9 activity as well as ADAM10, ADAM17, collagen deposition (Sirius red), hepatic stellate cell activation (α-SMA) and pCK+ reactive biliary cells were quantified. RESULTS: Only INT-787 significantly reduced serum ALT, IL-1ß and TGF-ß. A downregulation of RECK expression and protein levels observed in HFD groups (at 9 and 21 wks) was counteracted by both OCA and INT-787. HFD induced a significant increase in liver MMP-2 and MMP-9; OCA administration reduced both MMP-2 and MMP-9 while INT-787 markedly reduced MMP-2 expression. OCA and INT-787 reduced both ADAM10 and ADAM17 expression and number of pCK+ cells. INT-787 was superior to OCA in decreasing collagen deposition and α-SMA levels. CONCLUSION: INT-787 is superior to OCA in controlling specific cell types and clinically relevant anti-inflammatory and antifibrotic molecular mechanisms in NASH.

Intermittent mild cold stimulation alleviates cold stress-induced pulmonary fibrosis by inhibiting the TGF-ß1/Smad signaling pathway in broilers.

To investigate the potential protective effect of intermittent cold stimulation on lung tissues of broilers exposed to acute cold stress (ACS). A total of 384 one-day-old broilers were assigned to 4 experimental groups with 6 replicates of 16 birds each: control (CON) and ACS groups were reared at normal feeding temperature from d 1 to 42; cold treatment groups (CS3+ACS and CS9+ACS) were reared, respectively, at 3°C or 9°C for 5 h on alternate days below the CON group from d 15 to 35. Animals in CS3+ACS, CS9+ACS, and ACS groups were exposed at 10°C for 24 h on d 43. Subsequently, lung tissues were collected to perform histopathological examination and measurement of relevant indexes. The results showed that lung tissues in CS9+ACS and ACS groups exhibited increased inflammatory cell infiltrates and collagen deposition compared to the CON group, while this pathological phenomenon was less pronounced in the CS3+ACS group. Compared to CON group, H2O2 and MDA contents were increased, and the activities of antioxidant enzymes (CAT, SOD, GPx, T-AOC) were reduced in CS9+ACS and ACS group (P < 0.05); mRNA and protein levels of inhibitor of NF-κB, Smad7, matrix metallopeptidase (MMP)-2, MMP9, and antioxidant-related genes were downregulated, whereas mRNA and protein levels of genes related to NF-κB/NLRP3 pathway-regulated inflammation and TGF-ß1/Smad pathway-regulated fibrosis were upregulated in cold-stressed broilers (P < 0.05). mRNA levels of heme oxygenase-1, NAD(P)H quinone oxidoreductase-1, and MMP9 were increased in CS3+ACS group (P < 0.05). Moreover, the expression of most antioxidant-related genes was increased, and that of inflammation- and fibrosis-related genes was reduced in CS3+ACS group (P < 0.05). Therefore, cold stress caused oxidative stress and inflammation, leading to pulmonary fibrosis in broilers, whereas intermittent mild cold stimulation at 3°C below normal rearing temperature alleviated fibrosis by inhibiting the TGF-ß1/Smad pathway modulated by the Nrf2/HO-1 and NF-κB/NLRP3 signaling pathway. This study suggests that intermittent mild cold stimulation can be a potential strategy to reduce ACS-induced lung damage in broilers.

Matrix Metalloproteinase-9 inhibitors as therapeutic drugs for traumatic brain injury.

Traumatic brain injury (TBI) is one of the leading causes of morbidity and mortality among young adults and the elderly. In the United States, TBI is responsible for around 30 percent of all injuries brought on by injuries in general. Vasogenic cerebral edema due to blood-brain barrier (BBB) dysfunction and the associated elevation of intracranial pressure (ICP) are some of the major causes of secondary injuries following traumatic brain injury. Matrix metalloproteinase-9 (MMP-9) is a therapeutic target for being an enzyme that degrades the proteins that make up a part of the microvascular basal lamina as well as inter-endothelial tight junctions of the blood-brain barrier. MMP-9-mediated BBB dysfunctions and the compromise of the BBB is a major pathway that leads the development of vasogenic cerebral edema, elevation of ICP, poor cerebral perfusion and brain herniation following traumatic brain injury. That makes MMP-9 an effective therapeutic target and endogenous or exogenous MMP-9 inhibitors as therapeutic drugs for preventing secondary brain damage after traumatic brain injury. Although our understanding of the mechanisms that underlie the primary and secondary stages of damage following a TBI has significantly improved in recent years, such information has not yet resulted in the successful development of novel pharmacological treatment options for traumatic brain injury. Recent pre-clinical and/or clinical studies have demonstrated that there are several compounds with specific or non-specific MMP-9 inhibitory properties either directly binding and inhibiting MMP-9 or by indirectly inhibiting MMP-9, with potential as therapeutic agents for traumatic brain injury. This article reviews the efficacy of several such medications and potential agents that include endogenous and exogeneous compounds that are at various levels of research and development. MMP-9-based therapeutic drug development has enormous potential in the pharmacological treatment of cerebral edema and/or neuronal injury resulting from traumatic brain injury.

Andrographolide anti-proliferation and metastasis of hepatocellular carcinoma through LncRNA MIR22HG regulation.

Hepatocellular carcinoma (HCC) treatment is a major challenge. Although andrographolide (Andro) has an anti-proliferation effect on HCC, its underlying mechanism is not yet elucidated, and whether Andro can inhibit HCC metastasis has not been reported. The present study aimed to clarify whether Andro inhibits SK-Hep-1 cell proliferation and HCC metastasis, and the mechanisms. The results showed that Andro significantly reduced the survival of HCC cells and tumor weight and volume in tumor-bearing nude mice. Andro also triggered apoptosis of HCC cells and upregulated MIR22HG, Cleaved Caspase 9/7/3 expression levels, and downregulated BCL-2 mRNA, BCL-2 expression levels. Knockdown of MIR22HG or overexpression of HuR attenuated the effects of Andro on the signal transduction of mitochondrial apoptotic pathway and proliferation ability in HCC cells. Moreover, Andro significantly reduced the invasive ability of the cells and the level of HCC cell lung metastasis, upregulated miR-22-3p expression level and downregulated HMGB1 and MMP-9 expression levels. MIR22HG or miR-22-3p knockdown attenuated the effects of Andro on the signaling of HMGB1/MMP-9 pathway and invasive ability in HCC cells, while the overexpression of HMGB1 attenuated the inhibitory effects of Andro on the MMP-9 expression level and invasive ability in HCC cells. Thus, the regulation of MIR22HG-HuR/BCL-2 and MIR22HG/HMGB1 signaling pathways is involved in the anti-HCC proliferation and metastasis effects of Andro. This study provided a new pharmacological basis for Andro in HCC treatment and, for the first time, identified a natural product molecule capable of positively regulating MIR22HG, which has a robust biological function.

TIMP-1 Protects Tight Junctions of Brain Endothelial Cells From MMP-Mediated Degradation.

OBJECTIVE: The blood-brain barrier (BBB) plays a critical role in central nervous system homeostasis, and the integrity of BBB is disrupted in many neurodegenerative diseases. Matrix metalloproteinases (MMPs) degrade the tight junctions (TJs) of endothelial cells and basement membrane components essential to BBB integrity, which leads to increased BBB permeability and allows inflammatory cells and neurotoxic substances to enter the brain. Tissue inhibitors of metalloproteinases (TIMPs), endogenous inhibitors of MMPs, regulate MMP activity, thereby maintaining BBB integrity. METHODS: The disruptive impacts of MMP-3 and MMP-9 on BBB and protective effect of TIMP-1 were investigated in a simplified in vitro model of the BBB, which was generated using rat brain microvascular endothelial cells (RBMEC). The main features of BBB formation, including permeability and the trans-endothelial electrical resistance (TEER), were monitored over time after the addition of MMP-3 and MMP-9 and their complexes with TIMP-1 inhibitor. RESULTS: Our results indicated that MMP-3 and MMP-9 caused a dose-dependent disruption of the BBB, with 1.5 µM MMPs resulting in an over threefold increase in permeability, while TIMP-1 inhibition protected the integrity of the BBB model and recovered TEER and permeability of RBMECs. The disruption and recovery of tight junction proteins of RBMECs after MMP and TIMP treatment were also detected using fluorescent microscopy. CONCLUSION: MMP-9 and MMP-3 disrupt the BBB by degrading tight junctions in endothelial cells, and TIMP-1 could inhibit the disruptive effect of MMP-3 and MMP-9 by showing potential as therapeutic protein against MMP-related diseases where BBB disruption plays a role.

Effect of glycosaminoglycans on the structure and composition of articular cartilage and bone of broilers.

This study aimed to assess the influence of glycosaminoglycan (chondroitin and glucosamine sulfates) supplementation in the diet of broilers on the expression of matrix metallopeptidase 9 (MMP-9) and metallopeptidase inhibitor 2 (TIMP-2) genes, the synthesis of proteoglycans, collagen type II and chondrocytes, bone and cartilage macroscopy, bone mineral densitometry, bone breaking strength and mineral profile. A completely randomized design was carried out in a 3 × 3 factorial scheme (3 levels of chondroitin sulfate: 0.00, 0.05, and 0.10%; and 3 levels of glucosamine sulfate: 0.00, 0.15, and 0.30%), totaling 9 treatments. At 21 and 42 d of age, broilers were slaughtered, and tibias and femurs were collected for evaluation. There was an interaction (P < 0.05) of sulfates for the expression of MMP-9 and its inhibitor TIMP-2 in femur articular cartilage, as well as for the number of chondrocytes, collagen type II and proteoglycans in tibia articular cartilage, bone and cartilage macroscopy and mineral profile (P < 0.05), with better results obtained with the inclusion of chondroitin and/or glucosamine sulfates in the feed. In conclusion, chondroitin and glucosamine sulfates can be used in broiler diets in order to favor the development of the structure of the locomotor system (bones and joints), thus preventing locomotion problems.

The expression of tuftelin 1 as a new theranostic marker in early diagnosis and as a therapeutic target in hepatocellular carcinoma.

Currently, many challenges are associated with hepatocellular carcinoma (HCC) as the failure of early diagnosis, and the lack of effective therapy. This study aimed to investigate the possible role of tuftelin 1 (TUFT 1) in the early diagnosis of HCC and evaluate the potential contribution of the TUFT 1/Ca+2 /phosphinositol 3 kinase (PI3K) pathway in dantrolene sodium (Dan) therapeutic outcomes. The study was performed on two sets of rats, the staging (30 rats) and treatment sets (80 rats). HCC was induced by a single dose of diethylnitrosamine (DENA). The hepatic content of TUFT 1 protein was assayed via western blot and immunohistochemistry (IHC), while PI3K, vascular endothelial growth factor (VEGF), Cyclin D1, and matrix-metalloproteinase-9 (MMP-9) contents were assessed using enzyme-linked immunosorbent assay. Hepatic and serum calcium were measured colorimetrically. Furthermore, the nuclear proliferation marker, (Ki-67), (Kiel [Ki] where the antibody was produced in the University Department of Pathology and the original clone number is 67)-expression was assessed by IHC. TUFT 1/Ca+2 /PI3K signaling pathway was progressively activated in the 3 studied stages of HCC with subsequent upregulation of angiogenesis, cell cycle, and metastasis. More interestingly, Dan led to TUFT 1/Ca+2 /PI3K pathway disruption by diminution of the hepatic contents of TUFT 1, calcium, PI3K, VEGF, Cyclin D1, and MMP-9 in a dose-dependent pattern. TUFT 1 can serve as a theranostic biomarker in HCC. Moreover, Dan exerted an antineoplastic effect against HCC via the interruption of TUFT 1/Ca+2 /PI3K pathway.

Fenugreek Seed Extract Regulates Human Umbilical Vein Endothelial Cell Angiogenesis and Proliferation via the PI3K/Akt/Cyclin D1 Pathway.

The significance of angiogenesis in tumour progression has been widely documented. Hence, the identification of anti-angiogenic agents with fewer common side effects would be valuable in cancer therapy. In this study, we evaluated the anti-angiogenic and anti-proliferative effects of a hydro-alcoholic extract of fenugreek seed (HAEF) on human umbilical vein endothelial cells (HUVECs). Human umbilical vein endothelial cells were treated with various concentrations of HAEF and the half-maximal inhibitory concentration (IC50) value was estimated by using the MTT assay. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and matrix metalloproteinase enzyme (MMP-2 and MMP-9) gene expression profiles were evaluated by using quantitative RT-PCR (qRT-PCR). Moreover, MMP activities and PI3K, Akt and cyclin D1 protein expression levels were evaluated by gel zymography and Western blotting, respectively. HAEF reduced HUVEC viability, with an IC50 value of 200 µg/ml. The qRT-PCR results demonstrated that treatment with HAEF markedly reduced MMP-2/MMP-9, VEGF and bFGF gene expression, as compared to the control group. We also found that MMP-2/MMP-9 enzyme activity and PI3K/Akt/cyclin D1 protein expression were notably decreased in cells treated with HAEF. Our results suggest that HAEF can potentially inhibit angiogenesis, and also affect cellular proliferation by targeting the PI3K/Akt/cyclin D1 pathway. Thus, fenugreek seed extract merits further investigation as a source of compounds with anti-cancer properties.

Combined Pulsed Magnetic Field and Radiofrequency Electromagnetic Field Enhances MMP-9, Collagen-4, VEGF Synthesis to Improve Wound Healing Via Hif-1α/eNOS Pathway.

BACKGROUND: The blood supply of the tissue is very important in the acceleration of wound healing. Radiofrequency electromagnetic field (RF) and the pulsed magnetic field (PMF) increase vasodilation to contribute wound healing. The aim of this study was to evaluate the effects of RF and PMF on wound healing via hypoxia-inducible factor-1 alpha (Hif-1α)/endothelial nitric oxide synthase (eNOS) pathway. METHODS: Forty-eight rats were divided into 4 groups as sham (wound created only), PMF (27.12 MHz, 12 times a day at 30-min intervals), RF (0.5 mT, continuously) and PMF + RF groups. Wounds were created at 1.5 × 1.5 cm size to the dorsal region, and animals were put into unit. Six animals were killed on days 4 and 7; wound tissues were collected for histopathological, immunohistochemical as collagen-4, cytokeratin, matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF) staining and Hif-1α/eNOS/VEGF expressions. RESULTS: On day 4, in addition to increasing VEGF and MMP-9 stainings, connection between intact tissue and scar tissue which was stronger in the RF- and PMF-applied groups was observed. On day 7, epithelization started; inflammatory reaction decreased; collagen production, cytokeratin, VEGF and MMP-9 expression enhanced, especially in the RF + PMF applied group. eNOS, Hif-1α and VEGF expression levels were found to be significantly highest in both days of RF + PMF-applied group. CONCLUSIONS: This study revealed that both in vitro RF and PMF applications can cause notable changes in factors that are required for tissue repair on wound healing such as epithelization, connective tissue formation, collagen production and angiogenesis via vasodilatory Hif-1α/eNOS pathway and VEGF signaling. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Impact of house dust mite in vitiligo skin: environmental contribution to increased cutaneous immunity and melanocyte detachment.

BACKGROUND: Vitiligo is an autoimmune skin disorder characterized by loss of melanocytes. Protease-mediated disruption of junctions between keratinocytes and/or keratinocyte intrinsic dysfunction may directly contribute to melanocyte loss. House dust mite (HDM), an environmental allergen with potent protease activity, contributes to respiratory and gut disease but also to atopic dermatitis and rosacea. OBJECTIVES: To verify if HDM can contribute to melanocyte detachment in vitiligo and if so, by which mechanism(s). METHODS: Using primary human keratinocytes, human skin biopsies from healthy donors and patients with vitiligo, and 3D reconstructed human epidermis, we studied the effect of HDM on cutaneous immunity, tight and adherent junction expression and melanocyte detachment. RESULTS: HDM increased keratinocyte production of vitiligo-associated cytokines and chemokines and increased expression of toll-like receptor (TLR)-4. This was associated with increased in situ matrix-metalloproteinase (MMP)-9 activity, reduced cutaneous expression of adherent protein E-cadherin, increased soluble E-cadherin in culture supernatant and significantly increased number of suprabasal melanocytes in the skin. This effect was dose-dependent and driven by cysteine protease Der p1 and MMP-9. Selective MMP-9 inhibitor, Ab142180, restored E-cadherin expression and inhibited HDM-induced melanocyte detachment. Keratinocytes from patients with vitiligo were more sensitive to HDM-induced changes than healthy keratinocytes. All results were confirmed in a 3D model of healthy skin and in human skin biopsies. CONCLUSIONS: Our results highlight that environmental mite may act as an external source of pathogen-associated molecular pattern molecules in vitiligo and topical MMP-9 inhibitors may be useful therapeutic targets. Whether HDM contributes to the onset of flares in vitiligo remains to be tested in carefully controlled trials.

Kinesin family member 23 knockdown inhibits cell proliferation and epithelial-mesenchymal transition in esophageal carcinoma by inactivating the Wnt/ß-catenin pathway.

Kinesin family member 23 (KIF23) serves as a tumor-promoting gene with prognostic values in various tumors. However, the role of KIF23 in esophageal carcinoma (ESCA) progression is largely unknown. The overlapping differentially expressed genes (DEGs) in GSE12452, GSE17351, and GSE20347 datasets were identified via GEO2R tool and Venn diagram software. KIF23 expression was analyzed using GSE12452, GSE17351, and GSE20347 datasets, GEPIA database, and qRT-PCR. Cell proliferation was assessed by CCK-8 and EdU incorporation assays. Gene set enrichment analysis (GSEA) analysis was performed to investigate the pathways associated with the regulatory mechanisms of KIF23 in ESCA. The expression of E-cadherin, vimentin, N-cadherin, and matrix metalloproteinase-9 (MMP-9) and alternation of Wnt/ß-catenin pathway were detected by western blot analysis. We identified two overlapping upregulated DEGs, among which KIF23 was selected for subsequent experiments. KIF23 was overexpressed in ESCA samples and cells, and knockdown of KIF23 retarded cell proliferation in ESCA cells. Besides, KIF23 knockdown suppressed epithelial-mesenchymal transition (EMT) process in ESCA cells, as evidenced by the increase of E-cadherin expression and the reduction of vimentin, N-cadherin, and MMP-9 expression. GSEA analysis suggested that Wnt signaling pathway was the significant pathway related to KIF23. Moreover, we demonstrated that KIF23 silencing inhibited the Wnt/ß-catenin pathway in ESCA cells. Activation of Wnt/ß-catenin pathway by SKL2001 reversed the effects of KIF23 silencing on cell proliferation and EMT in ESCA cells. In conclusion, KIF23 knockdown inhibited the proliferation and EMT in ESCA cells through blockage of Wnt/ß-catenin pathway.

Synovial monocytes from psoriatic and rheumatoid arthritis patients are modulated differently by TNF inhibitors and glucocorticoids: an ex-vivo study.

OBJECTIVES: Synovial monocytes (expressing CD14+CD16+) affect pro-inflammatory responses in the synovium microenvironment of psoriatic arthritis (PsA) and rheumatoid arthritis (RA). The effect of various drugs on those cells was evaluated. METHODS: Synovial fluid mononuclear cells (SFMCs) from PsA (n=29) and RA (n=11) patients were cultured with biologics or glucocorticoids (GCs). CD14+CD16+ cells were analysed by flow cytometry. TNF secretion was assessed by ELISA and changes in cytokine and matrix metalloproteinase-9 (MMP-9) mRNA by qPCR. RESULTS: TNF inhibitors (i) [adalimumab (ADA) and infliximab (IFX)] significantly reduced the %CD14+CD16+ cells (p<0.04 and p<0.02, respectively) compared to IL-17Ai, IL-12/23i, and GCs in PsA patients' SFMCs. Similarly, those TNFi reduced the %CD14+CD16+ cells (p<0.05 and p<0.02, respectively) compared to IL-6Ri, CD20i and GCs in RA patients' SFMCs. TNFi (ADA p<0.01, IFX p=0.0003), and GCs (p<0.05) reduced TNF levels in PsA patients SFMCs supernatants. IFX down-regulated IL-1ß mRNA (p<0.005) while GCs betamethasone (BET) (p<0.01) and methylprednisolone acetate (MPA) (p<0.005) led to IL-1ß up-regulation. IFX down-regulated IL-8 and MMP-9 (p<0.01) and up-regulated IL-10 (p<0.005), and GCs did so to a greater extent (for IL-8, BET p<0.0001 and MPA p<0.005, for MMP-9, BET and MPA p<0.0001 and for IL-10, BET and MPA p<0.0001). CONCLUSIONS: TNFi but not GCs reduced the inflammatory monocytes. Both TNFi and GCs inhibited TNF secretion but differently modulated IL-1ß, IL-8, MMP-9 and IL-10 gene expression. Our data point to TNFi as a modulator of synovial monocytes.

Impact of Vitamin D3 on Carbonyl-Oxidative Stress and Matrix Metalloproteinases after Acute Intracerebral Hemorrhage in Rats with Type 2 Diabetes Mellitus.

INTRODUCTION: Diabetes mellitus is associated with the development of carbonyl-oxidative stress (COS) and an increased risk of a cerebral hemorrhage. Vitamin D3 is considered an additional drug to have an impact on COS and proteolysis in the extracellular matrix. OBJECTIVE: The study aimed to evaluate the impact of D3 on the COS-markers and matrix metalloproteinases MMP2/MMP9 activity after acute intracerebral hemorrhage (ICH) in rats with experimental type 2 diabetes mellitus (Т2DM) compared to metformin (Met). METHODS: T2DM was induced in rats via the intraperitoneal injection of streptozotocin (STZ) and nicotinamide (NA), ICH - by microinjection of bacterial collagenase into the striatum. Rats were randomized into five groups: 1 - intact animals (n = 8), 2 - T2DM (n = 9); 3 - T2DM+ICH (n = 7); 4 - T2DM+ICH+Met (n = 7); 5 - T2DM+ICH+D3 (n = 7). Blood glucose, glycated hemoglobin, and oral glucose tolerance test (OGTT) were assessed using commercial kits. Advanced oxidation protein products (AOPP), protein carbonyls (PC370/430), and ischemia-modified albumin (IMA) were measured by spectrophotometry, advanced glycation end products (AGEs) by quantitative fluorescence, and matrix metalloproteinases MMP2/9 by gelatin zymography. RESULTS: D3 does not significantly affect the glucose level and OGTT in rats with T2DM+ICH. However, it reduces AOPP, PC, and AGEs, thus reducing the COS index. In contrast, the activity of proMMP9 increases after D3 administration. These effects of D3 have been reported to be stronger and sometimes opposite to those of metformin. CONCLUSION: D3 supplementation may decrease the negative consequences of a cerebral hemorrhage in T2DM by reducing COS and preventing the accumulation of COS-modified proteins in the brain by regulating the expression and activity of MMP9.

A bio-polymeric scaffold incorporated with p-Coumaric acid enhances diabetic wound healing by modulating MMP-9 and TGF-ß3 expression.

Diabetic wounds lead to severe health complications as the tissue regeneration process fails predominantly due to prolonged inflammation, reactive oxygen species generation, and imbalance in collagen turnover. Modern wound dressings that can aid in wound management thus improving the public healthcare system, is the present need. This study aims to fabricate an effective wound dressing using plant polyphenol to treat chronic wounds as polyphenols possess excellent wound-healing ability. The collagen scaffold enriched with the polyphenol, p-Coumaric acid, was fabricated by freeze-drying method (Col-OxP3-Ca) and examined for its wound-healing ability by in vitro and in vivo analyses. Col-OxP3-Ca scaffold exhibited 85% antioxidant activity, biocompatibility in fibroblast cells, enhanced cell proliferation and migration rate. The diabetic excision wound treated with Col-OxP3-Ca scaffold healed within 21 days and a well-developed epidermis, blood vessels, hair follicle formation, fewer inflammatory cells and collagen deposition was observed in histological analysis. The immunohistochemical results depicted the enhanced expression of TGF-ß3 and lessened expression of the MMP-9 in Col-OxP3-Ca scaffold treatment group. p-Coumaric acid shortened the inflammatory stage, enhanced angiogenesis, tissue regeneration and balanced collagen turnover during healing. From this, we can accomplish that the Col-OxP3-Ca wound dressing could be an outstanding alternative to treat chronic wounds.

In vitro synergistic effect of hesperidin and doxorubicin downregulates epithelial-mesenchymal transition in highly metastatic breast cancer cells.

BACKGROUND: We previously reported that in highly metastatic breast cancer cells, doxorubicin (DOX) at non-toxic concentrations promoted cell migration and invasion. Hesperidin (30, 5, 9-dihydroxy-40-methoxy-7-orutinosyl flavanone) is a flavonoid glycoside isolated from citrus/lemon plant that possesses a cytotoxic effect in several cancer cells. In this study, we investigate whether DOX efficacy is enhanced by hesperidin (Hsd) and the molecular pathway involved in highly metastatic breast cancer, 4T1. METHODS: Combined cytotoxicity of Hsd and DOX was evaluated with MTT assay and was analyzed using Chou-Talalay's method. To better understand the underlying mechanism, several factors, including apoptosis and cell cycle arrest were analyzed by flow cytometry. In addition, antimigration activity was evaluated by scratch wound healing assay, MMP-9 expression by ELISA and gelatin zymography, and Rac-1 protein level using western blot. The data on survival rate and expression level of MMP-9 and Rac-1 were obtained from Gene Expression OMNIBUS (GEO). RESULTS: Under MTT assay, Hsd showed a cytotoxic effect in a concentration-dependent manner with an IC50 value of 284 µM on 4T1 cells. Hsd synergistically enhanced the cytotoxic effect of DOX which seemed to correlate with an increase in apoptotic cell death, G2/M cell cycle arrest and blocked the migration of 4T1 cells. At 10 nM, doxorubicin induced lamellipodia formation, and increased the level of Rac-1 and metalloproteinase-9 (MMP-9) expression. Interestingly, combined treatment of DOX and Hsd dramatically downregulated the expression of MMP-9 and Rac-1. These results indicated that Hsd block the cell migration induced by DOX under in vitro studies. CONCLUSION: These findings strongly suggest that Hsd possesses a potential synergistic effect that can be developed to enhance the anticancer efficacy of DOX and reduce the risks of chemotherapy use in highly metastatic breast cancer.

Citral Modulates MMP-2 and MMP-9 Activities on Healing of Gastric Ulcers Associated with High-Fat Diet-Induced Obesity.

Obesity causes low-grade inflammation that results in the development of comorbidities. In people with obesity, exacerbation of gastric lesion severity and delayed healing may aggravate gastric mucosal lesions. Accordingly, we aimed to evaluate the citral effects on gastric lesion healing in eutrophic and obese animals. C57Bl/6 male mice were divided into two groups: animals fed a standard diet (SD) or high-fat diet (HFD) for 12 weeks. Gastric ulcers were induced using acetic acid (80%) in both groups. Citral (25, 100, or 300 mg/kg) was administered orally for 3 or 10 days. A vehicle-treated negative control (1% Tween 80, 10 mL/kg) and lansoprazole-treated (30 mg/kg) were also established. Lesions were macroscopically examined by quantifying regenerated tissue and ulcer areas. Matrix metalloproteinases (MMP-2 and -9) were analyzed by zymography. The ulcer base area between the two examined periods was significantly reduced in HFD 100 and 300 mg/kg citral-treated animals. In the 100 mg/kg citral-treated group, healing progression was accompanied by reduced MMP-9 activity. Accordingly, HFD could alter MMP-9 activity, delaying the initial healing phase. Although macroscopic changes were undetectable, 10-day treatment with 100 mg/kg citral exhibited improved scar tissue progression in obese animals, with reduced MMP-9 activity and modulation of MMP-2 activation.

Expression of HMGB1-TLR4 in Placentas from Preeclamptic Pregnancies and Its Effect on Proliferation and Invasion of HTR-8/SVneo Cells.

OBJECTIVES: Placental inflammation possibly underlies preeclampsia (PE) pathogenesis. This study aimed to investigate the expression of the high mobility box group 1 (HMGB1)-toll-like receptor 4 (TLR4) signalling pathway in preeclamptic placentas and determine whether HMGB1 regulates the biological behaviour of trophoblasts in vitro. DESIGN: Placental biopsies were taken from 30 preeclamptic patients and 30 normotensive controls. In vitro experiments were carried out in HTR-8/SVneo human trophoblast cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: HMGB1, TLR4, and nuclear factor kappa B (NF-κB) mRNA and protein were quantified to compare their expression in human placentas from preeclamptic and normotensive pregnancies. HTR-8/SVneo cells were stimulated with HMGB1 (50-400 µg/L) for 6-48 h, and proliferation and invasion of HTR-8/SVneo cells were measured via Cell Counting Kit-8 and transwell assays. HTR-8/SVneo cells were also transfected with HMGB1 and TLR4 small interfering RNA (siRNA) to investigate the effect of knocking down these proteins. The mRNA and protein expression of TLR4, NF-κB, and matrix metalloproteinase 9 (MMP-9) were determined using quantitative real-time PCR and Western blotting, respectively. Data were analysed with either a t-test or one-way analysis of variance. RESULTS: The mRNA and protein levels of HMGB1, TLR4, and NF-κB were significantly higher in the placentas from preeclamptic pregnancies than from normal pregnancies (p < 0.05). HMGB1 stimulation (at concentrations up to 200 µg/L) of HTR-8/SVneo cells significantly increased invasion and proliferation over time. However, at an HMGB1 stimulation concentration of 400 µg/L, the invasion and proliferation ability of HTR-8/SVneo cells decreased. Compared to controls, mRNA and protein expression levels of TLR4, NF-κB, and MMP-9 increased (mRNA level fold change: 1.460, 1.921, 1.667; protein level fold change: 1.600, 1.750, 2.047) when stimulated with HMGB1 (p < 0.05) but decreased when HMGB1 was knocked down (p < 0.05). TLR4 siRNA transfection combined with HMGB1 stimulation reduced the mRNA (fold change: 0.451) and protein (fold change: 0.289) expression of TLR4 (p < 0.05), while NF-κB and MMP-9 were unaffected (p > 0.05). LIMITATIONS: Only one trophoblast cell line was used in this study, and the findings were not confirmed in animal studies. CONCLUSIONS: This study explored the pathogenesis of PE from two aspects: inflammation and trophoblast invasion. The overexpression of HMGB1 in placentas from preeclamptic pregnancies suggests this protein may be involved in PE pathogenesis. In vitro, HMGB1 was found to regulate the proliferation and invasion of HTR-8/SVneo cells by activating the TLR4-NF-κB-MMP-9 pathway. These findings have implications for targeting HMGB1 could be a therapeutic strategy for treating PE. In the future, we will further verify this in vivo and in other trophoblast cell lines, further exploring the molecular interactions of the pathway.