This study aims to investigate the wound-healing effectiveness of the phenolic compound, naringin, both in vitro and in vivo. Male mice were shaved on their dorsal skin under isoflurane, a biopsy punch was made in four symmetrical circular resection windows (6 mm) to induce a wound. These excision wounds were used to study the topical effects of naringin in terms of various biochemical, molecular, and histological parameters. We observed a significant recovery in the wound area. Increased levels of MMP-2, 9, 14, TIMP-2, VEGF-A, and VEGF-R1 were induced by naringin in the HaCaT cells. The time course experiments further revealed that levels of VEGF-A and B increased within 36 h; whereas levels of VEGF-C decreased. In line with this, VEGF-R3 levels, but not VEGF-R1 and 2 levels, increased soon after stimulation; although the increase subsided after 36 h. Additionally, naringin cream upregulated wound healing in vitro. The blockage of VEGF by Bevacizumab abolished the function of naringin cream on cell migration. Histological alterations in the wounded skin were restored by naringin cream, which accelerated wound healing via upregulated expression of growth factors (VEGF-A, B, and C and VEGF-R3), and thus increased MMP-2, 9, 14 expressions.

Gallic acid is a trihydroxybenzoic acid found in natural herbal plants. Gallic acid has been reported to inhibit the migration and invasive capability of various cancers. Little is known about the underlying mechanisms of invasion responsible for cancer metastasis via gallic acid. The present study was intended to investigate the anti-invasive effect of gallic acid on human nasopharyngeal carcinoma cells (NPC-BM1) and its related mechanism. Gallic acid inhibited the invasion of NPC-BM1 cells dose- and time-dependently without significant cytotoxic effect. Affymetrix oligonucleotide microarray analysis revealed matrix metalloproteinase-1 (MMP-1) as the most down-regulated gene in NPC-BM1 cells by gallic acid. The cytosolic and secreted MMP-1 levels were both found to be inhibited by gallic acid as demonstrated by western blot analysis and ELISA respectively. The mRNA expression and transcription of MMP-1 gene was also down-regulated as determined by RT/real-time PCR and promoter activity assay. The expression of two major transcription binding factors in the MMP-1 promoter, AP-1 and ETS-1, were demonstrated to be reduced by gallic acid in NPC-BM1 cells. The effect of gallic acid was associated with the inhibition of p38 MAPK signaling pathway. In addition, gallic acid enhanced the gene expression of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) which further suppressed the MMP-1 activity. These findings may be useful to develop a novel chemotherapeutic agent to inhibit the metastasis of nasopharyngeal cancer.

Introduction: Transglutaminase 2 (TG2), a protein crosslinking enzyme with multiple biochemical functions, has been connected to various inflammatory processes. In this study, the involvement of TG2 in monosodium urate (MSU) crystal-induced inflammation was studied. Methods: Immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) were performed to detect TG2 expression in synovial fluid mononuclear cells (SFMCs) and synovial tissue from patients with gouty arthritis. MSU crystal-exposed RAW264.7 mouse macrophages were analyzed for interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), transforming growth factor β1 (TGF-β1) and TG2 expression by RT-PCR and enzyme-linked immunosorbent assay (ELISA). TG2 small interfering (si)-RNA-mediated silencing and overexpression in RAW264.7 cells were used to evaluate the involvement of TG2 in resolving MSU crystal-induced inflammation. The role of metastatic tumor antigen 1 (MTA1), a master chromatin modifier, was investigated by MTA1 si-RNA-mediated knockdown. In addition, the inflammatory responses were followed in wild type and TG2 null mice after being challenged with MSU crystals in an in vivo peritonitis model. Results: TG2 expression was up-regulated in the synovium tissue and SFMCs from patients with gouty arthritis. The levels of MTA1, TG2, TGF-β1, IL-1β and TNF-α mRNAs were consistently increased in MSU crystal-stimulated RAW264.7 cells. si-MTA1 impaired the basal, as well as the MSU crystal-induced expression of TG2 and TGF-β1, but increased that of IL-1β and TNF-α. TG2 overexpression dramatically suppressed MSU crystal-induced IL-1β and TNF-α, but significantly enhanced the TGF-β1 production. Neutralizing TGF-β antibodies or inhibition of the crosslinking activity of TG2 attenuated these effects. On the contrary, loss of TG2 resulted in a reduced TGF-β, but in an increased IL-1β and TNF-α production in MSU crystal-stimulated RAW264.7 cells and mouse embryonic fibroblasts (MEFs). MSU crystal-stimulated IL-1β production was Janus kinase 2 (JAK2)-signaling dependent and TG2-induced TGF-β suppressed the activity of it. Finally, TG2-deficient mice exhibited hyper inflammatory responses after being challenged with MSU crystals in an in vivo peritonitis model. Conclusions: These findings reveal an inherent regulatory role of the MTA1-TG2 pathway in the self-limitation of MSU crystal-induced inflammation via positively regulating the levels of active TGF-β1 in macrophages that opposes the MSU crystal-induced JAK2-dependent pro-inflammatory cytokine formation.

Discovering how to regulate mitochondrial function to reduce cancer growth holds great potential for future cancer therapy development. Here we explore the effects of cryptotanshinone (CPT), a natural product derived from Salvia miltiorrhiza, on mitochondria of osteosarcoma (OS) both in vitro and in vivo, and further elucidate the underlying molecular mechanisms. Cytotoxicity in the CPT treated OS cells was analyzed by flow cytometry, CCK8, TUNEL assay and colony formation assays. Flow cytometric analysis was performed to evaluate the effect of CPT on cell cycle of OS cells. Mitochondrial morphology was examined by staining with the mitochondrial membrane potential -sensitive fluorochrome, MitoTracker Red (CMXRos). Immunoblotting, confocal-immunofluorescence staining, co-immunoprecipitation were used to examine the expression and interaction between CPT-mediated Drp1 and Bax. Finally, the synergistic effect of CPT on OS cells was validated using a mouse xenograft tumor model. In this study, we found CPT treatment induced S-phase arrest, apoptosis, and mitochondrial fragmentation in OS cells. CPT also effectively activated caspase-dependent apoptosis, which could be blocked by pan-caspase inhibitor Z-VAD-FMK. Moreover, we herein provide evidence that treatment with CPT resulted in mitochondrial fragmentation, which is mediated by dynamin-related protein 1 (Drp1), a key mediator of mitochondrial fission. Pursuing this observation, downregulation of Drp1 via silencing RNA could abrogate the induction of apoptosis and mitochondrial fragmentation induced by CPT. Finally, we demonstrate that CPT induced Drp1, which interacted directly with Bcl-2-associated X protein (Bax), which contributed to driving Bax translocation from the cytosol to the mitochondria. Our findings offer insight into the crosstalk between mitochondrial fragmentation and inhibition of osteosarcoma cell growth in response to CPT.

Background: To assess the effects of Glycine tomentella Hayata (GTH), a traditional herbal medicine for treatment of rheumatic diseases on the expression of the proinflammatory cytokines and on the clearance of apoptotic cells by macrophages. Methods: RAW264.7 cells were cultured with lipopolysaccharide (LPS) in the presence or absence of ethanol extract of GTH. The expression of proinflammatory cytokines IL-1β, IL-6, and TNF-α, and inducible nitric oxide synthase (iNOS) and transglutaminase 2 (TG2) were assayed by reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Matrix metalloproteinase (MMP)-2 and MMP-9 were assayed by gelatin zymography. For detecting uptake of apoptotic cells, RAW264.7 cells were cultured with carboxyfluorescein diacetate (CFDA)-stained apoptotic cells and assayed by flow cytometry. Results: The major components of GTH analyzed by high-performance liquid chromatography (HPLC) chromatogram were daidzein (42.5%), epicatechin (28.8%), and naringin (9.4%). GTH treatment inhibited the expression of proinflammatory cytokines IL-1β, IL-6 and MMP-9 but did not affect the expression of TNF-α and iNOS. GTH significantly enhanced the expression of TG2 and the clearance of apoptotic cells by RAW264.7 macrophages. Conclusions: GTH inhibits proinflammatory cytokine secretion and MMP-9 activity, enhances apoptotic cell uptake and up-regulates TG2 expression. Our data show that GTH might have beneficial effects on rheumatic diseases.

Hepatocellular carcinoma (HCC) is a primary malignancy of the liver. In basic studies, the regulation of autophagy has offered promising results for HCC treatment. This study aimed to address the question of whether amiodarone can improve survival rates in HCC patients associated with autophagy. Using datasets from the National Health Insurance Research Database, we enrolled patients over 18 years of age that had been diagnosed with HCC between January 1997 and December 2010. Amiodarone and non-amiodarone users were matched at a 1:1 frequency, according to all variables. Additionally, HepG2 cells treated with amiodarone were evaluated by cell viability and autophagic change. Autophagic signaling was examined by immunoblotting and tissue array immunohistochemistry. Of the 10,946 patients diagnosed with HCC, each cohort included 221 patients after 1:1 propensity score matching. The median survival was 36.70 months for the amiodarone users, and 24.48 months for the non-amiodarone users. After adjusting for age, gender, comorbidities and treatment, amiodarone users had a significantly lower risk of mortality. Amiodarone users also demonstrated an improved 3-year survival rate. Furthermore, amiodarone treatment-induced autophagy in HepG2 cells was demonstrated by autophagosome formation associated with increasing LC3B-II, P62, and Beclin-1 expression. Autophagic flux also increased following amiodarone treatment with bafilomycin A1. SiRNA of LC3B knocked down endogenous LC3B formation and restored HepG2 cell viability. This study provides epidemiologic evidence that amiodarone via autophagic degradation machinery may offer survival benefits for HCC patients with a history of arrhythmia. Further randomized, blinded, and placebo-controlled trials are warranted for patients with HCC.