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Hui Ma,1,2 Ping Kuen Lam,3 Wing Sum Siu,1,2 Cindy See Wai Tong,3 Kin Ki Yan Lo,3 Chi Man Koon,1,2 Xiao Xiao Wu,1,2 Xiang Li,1,2 Wen Cheng,1,2 Wai Ting Shum,1,2 Ping Chung Leung1,2 1Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, Special Administrative Region of China; 2State Key Laboratory of Research on Bioactivities and Clinical Applications of Medicinal Plants, The Chinese University of Hong Kong, Shatin, Hong Kong, Special Administrative Region of China; 3Chow Tai Fook-Cheng Yu Tung Surgical Stem Cell Research Center, Department of Surgery, The Chinese University of Hong Kong, Shatin, Hong Kong, Special Administrative Region of ChinaCorrespondence: Ping Chung LeungInstitute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, Special Administrative Region of ChinaTel +85222528868Email pingcleung@cuhk.edu.hkIntroduction: The biological role of mesenchymal stem cells (MSCs) in wound healing has been demonstrated. However, there were limited studies on the healing effect of secretome which consists of many biological factors secreted by MSCs. In this study, we aimed to compare the therapeutic effects of secretome with MSCs on facilitating wound healing.Methods: Green fluorescent protein labelled adipose-derived MSCs (GFP-ADMSCs) or secretome was injected in the full-thickness skin excision model on SD rats. The wound healing process was evaluated by calculating the healing rate and the histological examinations on skin biopsy. The cell viability, proliferation and mobility of the rat dermal fibroblasts were compared after different treatments. The inflammatory response in macrophages was indicated by the level of nitric oxide (NO) and inflammatory cytokines through NO assay and ELISA.Results: On day 5 and day 14, both MSCs and secretome accelerated the wound healing, secretome further enhanced the process. GFP-MSCs were detected 10 days after transplantation. The level of IL-6 and TNF-α in blood was reduced after MSCs and secretome treatments. The expressions of VEGF and PCNA were increased after treatment, higher intensity of VEGF was observed in secretome-injected tissue. The concentrations of total protein and VEGF in secretome were 2.2 ± 0.5 mg/mL and 882.0 ± 72.7 pg/mL, respectively. The cell viability and proliferation of FR were promoted significantly after the treatment. The scratch test showed that secretome accelerated the wound healing speed. Secretome reduced the metabolism of macrophages remarkably, but it did not decrease the level of macrophage-secreted NO. The expression of the pro-inflammatory cytokines (IL-6, MCP-1 and TNF-α) was downregulated significantly.Conclusion: Our study indicated both MSCs and MSCs-derived secretome enhanced the wound healing process in early phase. Secretome further promoted the healing effects through promoting the fibroblast proliferation and migration and suppressing the inflammatory response.Keywords: MSCs, secretome, wound healing, local injection, skin

Legionella pneumophila can cause pneumonia, leading to severe acute respiratory distress syndrome (ARDS). Because of its harsh growth requirements, limited detection methods, and non-specific clinical manifestations, diagnosing Legionella pneumonia remains still challenging. Metagenomic next-generation sequencing (mNGS) technology has increased the rate of detection of Legionella. This study describes a patient who rapidly progressed to severe ARDS during the early stage of infection and was treated with extracorporeal membrane oxygenation (ECMO). Although his bronchoalveolar lavage fluid (BALF) was negative for infection and his serum was negative for anti-Legionella antibody, mNGS of his BALF and blood showed only the presence of Legionella pneumophila (blood mNGS reads 229, BALF reads 656). After antibiotic treatment and weaning from ECMO, however, he developed a secondary Aspergillus and Klebsiella pneumoniae infection as shown by mNGS. Mechanical ventilation and antibiotic treatment were effective. A search of PubMed showed few reports of secondary Aspergillus infections after Legionella infection. Severe pneumonia caused by any type of pathogenic bacteria may be followed by Aspergillus infection, sometimes during extremely early stages of infection. Patients with severe pneumonia caused by Legionella infection should undergo early screening for secondary infections using methods such as mNGS, enabling early and precise treatment, thereby simplifying the use of antibiotics and improving patient prognosis.

HER2-positive breast cancer accounts for 15–20% in breast cancer and 50% of the metastatic HER2-positive breast cancer patients died of central nervous system progression. The present study investigated the effects of actein (a natural cycloartane triterpene) on cells adhesion, migration, proliferation and matrix degradation, and its underlying mechanism in HER2-positive breast cancer cells. The in vivo effect of actein on tumor growth and metastasis in MDA-MB-361 tumor-bearing mice as well as the anti-brain metastasis in tail vein injection mice model were also investigated. Our results showed that actein inhibited HER2-positive breast cancer cells viability, proliferation and migration. Actein also induced MDA-MB-361 cells G1 phase arrest and inhibited the expressions of cyclins and cyclin-dependent kinases. For intracellular mechanisms, actein inhibited the expressions of molecules in AKT/mTOR and Ras/Raf/MAPK signaling pathways. Furthermore, actein (15 mg/kg) was shown to exhibit anti-tumor and anti-metastatic activities in MDA-MB-361 breast tumor-bearing mice, and reduced brain metastasis in tail vein injection mice model. All these findings strongly suggested that actein is a potential anti-metastatic agent for HER2-positive breast cancer.

Yuanyuan Han,1,2 Xuxia Wang,2,3 Dan Ma,1,2 Xiaoxiao Wu,1,2 Panpan Yang,1,2 Jun Zhang1,2 1Department of Orthodontics, Faculty of Stomatology, Shandong University, Jinan, 2Shandong Provincial Key Laboratory of Oral Tissue Regeneration, School of Stomatology, Shandong University, Jinan, 3Department of Oral and Maxillofacial Surgery, Faculty of Stomatology, Shandong University, Jinan, China Objectives: This study was performed to investigate the effects and mechanism of ipriflavone (IP) on the proliferation and osteoblastic differentiation of periodontal ligament cells in vitro and periodontal tissue remodeling following orthodontic tooth movement (OTM) in vivo. Materials and methods: Human periodontal ligament cells (hPDLCs) were cultured in vitro and cell counting kit-8, alkaline phosphatase (ALP) activity assay, plate clone formation assay, and alizarin red staining were used to test proliferation and osteogenic differentiation of hPDLCs. What is more, the expression of ALP, Runx2, and GPR30 was examined by real-time polymerase chain reaction and Western blot. To find out if PI3K/AKT signaling pathway was involved in theprocess, AKT and p-AKT were examined by Western blot. LY294002 (PI3K signaling pathway inhibitor) and small interfering RNA targeting GPR30 mRNA (siGPR30) were used to verify the function of GPR30-mediated PI3K/AKT pathway in this process. Twenty-four male Wistar rats were randomized into 2 groups, the control group with force application and the IP group with force application plus IP. Morphological changes in the periodontal tissue between roots of teeth were investigated using hematoxylin and eosin (HE) staining and bone morphogenetic protein-2 was detected to assess bone remodeling by immunohistochemical staining. Results: In vitro, 10-7 M IP was selected significantly promoting proliferation, ALP activity, colony forming efficiency, and mineral deposition (P

Objective To discuss the effect of miR-183 on osteoblast differentiation in the osteoporosis progression via targeting Smad4. Methods Osteoporosis models were constructed on ovariectomized (OVX) mice to determine the expression of miR-183 and Smad4. Then, MC3T3-E1 cells and primary osteoblasts were divided into Mock, miR-control, miR-183 mimic, miR-183 inhibitor, siSmad4 and miR-183 inhibitor + siSmad4 groups. Alkaline phosphatase (ALP) staining were performed to determine ALP activity, alizarin red staining to evaluate the calcium deposit, while qRT-PCR and Western blotting were used to determine the expression of related molecules. Besides, MC3T3-E1 cells transfected with miR-control or miR-183 mimic were cultured with or without TGF-β1 to verify whether miR-183 regulates the TGF-β signaling pathway. Results MiR-183 was up-regulated with decreased Smad4 in the femur of OVX mice, and dual luciferase reporter gene assay showed that Smad4 was a target of miR-183. As compared to Mock group, MC3T3-E1 cells and primary osteoblasts in the miR-183 mimic group and siSmad4 group had significant reductions of OCN, OPN, Runx2 and Osx, as well as decreased ALP activity and calcium deposit. Contrarily, miR-183 and Smad4 were up-regulated and down-regulated respectively. However, cells in the miR-183 inhibitor group manifested the opposite changes. Besides, osteoblast differentiation in the miR-183 inhibitor + siSmad4 group was weakened evidently when compared to miR-183 inhibitor group. Pathway analysis indicated that miR-183 regulated osteogenic differentiation via TGF-β signaling pathway. Conclusion MiR-183 was up-regulated in osteoporosis, and miR-183 overexpression can inhibit osteoblast differentiation by targetedly down-regulating TGF-β pathway member Smad4 to trigger osteoporosis.

Background Atopic dermatitis (AD) is a chronic relapsing inflammatory and pruritic skin disease, affecting 10-20% of the population worldwide. Paeonia suffruticosa Andrews (Paeoniaceae) (Cortex Moutan) and Mentha haplocalyx Briq. (Labiatae) (Herba Menthae) have shown beneficial effects on AD. Calendula officinalis L. (Asteraceae) is commonly used for treating skin rashes and wounds. Purpose In the present study, a three-herbs formula including Cortex Moutan and Herba Menthae, together with C. officinalis at 1:1:1 weight ratio was used as a topical agent and its therapeutic effects on AD was investigated. Methods In vitro effects of individual herbs and three-herbs formula (0.125–1 mg/ml) were examined using cytokine release assay on human mast HMC-1 cells, inflammation test on murine macrophage RAW cells and human keratinocyte (HaCaT) cells, and migration scratch assay on human umbilical vein endothelial cells (HUVEC). The contributing functional pathway of three-herbs formula in AD was explored using Western Blot assay in HMC-1 cells. Oxazolone-induced AD-like mice model was also used to investigate the in vivo therapeutic effect of the topical application of the three-herbs formula. Results Herba Menthae, Cortex Moutan, and three-herbs formula significantly reduced the production of IL-6 and tumor necrosis factor (TNF)-α in HMC-1 cells, inhibited the expression of IL-6, IL-8 and CCL2 in TNF-α/IFN-γ stimulated HaCaT cells, and suppressed the lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 cells. Moreover, Herba Menthae and three-herbs formula significantly suppressed CCL2 and TNF-α production in LPS-induced RAW 264.7 cells. C. officinalis and three-herbs formula promoted wound healing in HUVEC. For intracellular mechanisms, three-herbs formula inhibited the expressions of molecules in STAT1 and STAT3-dependent pathways. In vivo model showed that topical application of three-herbs formula on challenged ear reduced ear swelling and mice scratching frequencies. H&E and toluidine blue staining of the challenged ear tissue demonstrated that three-herbs formula reduced the epidermal thickness and mast cell infiltration, respectively. Conclusion The three-herbs formula of Cortex Moutan, Herba Menthae and C. officinalis at 1:1:1 (w/w) exhibited anti-inflammatory effect and promotion of cell migration in vitro. It also alleviated ear redness, swelling, epidermal thickness and inflammation of the OXA-induced AD mice. These findings suggest a potential beneficial role of the topical application of the three-herbs formula for treatment of AD.

Background: Tumor necrosis factor-alpha (TNF-α) is a major proinflammatory cytokine that has been posited to be involved in the development of chronic pancreatitis (CP). Several studies have been c...

// Cong Wang 1, * , Ping Ruan 1, * , Ying Zhao 1 , Xiaomin Li 1 , Jun Wang 1 , Xiaoxiao Wu 1 , Tong Liu 1 , Shasha Wang 1 , Jiuzhou Hou 1 , Wei Li 1 , Qian Li 2 , Jinghua Li 2 , Fujun Dai 2 , Dong Fang 1 , Chaojie Wang 2 , Songqiang Xie 1 1 Institute of Chemical Biology, College of Pharmacy, Henan University, Kaifeng, 475004, China 2 The Key Laboratory of Natural Medicine and Immuno-Engineering, Henan University, Kaifeng, 475004, China * These authors have contributed equally to this work Correspondence to: Dong Fang, email: emailfangdong@163.com Chaojie Wang, email: wcjsxq@henu.edu.cn Songqiang Xie, email: xiesq@henu.edu.cn Keywords: SSAT, polyamine, metastasis, AKT, β-catenin Received: May 31, 2016 Accepted: November 12, 2016 Published: November 25, 2016 ABSTRACT Hepatocellular carcinoma (HCC) and colorectal cancer (CRC) are among the most common cancers across the world. Therefore, identifying the potential molecular mechanisms that promote HCC and CRC progression and metastasis are urgently needed. Spermidine/spermine N 1 -acetyltransferase (SSAT) is a catabolic enzyme that acetylates the high-order polyamines spermine and spermidine, thus decreasing the cellular content of polyamines. Several publications have suggested that depletion of intracellular polyamines inhibited tumor progression and metastasis in various cancer cells. However, whether and how SSAT regulates cell growth, migration and invasion in hepatocellular and colorectal carcinoma cells remains unclear. In this study, depletion of polyamines mediated by SSAT not only attenuated the tumor cell proliferation but also dramatically inhibited cell migration and invasion in hepatocellular and colorectal carcinoma cells. Subsequent investigations revealed introduction of SSAT into HepG2, SMMC7721 hepatocellular carcinoma cells and HCT116 colorectal carcinoma cells significantly suppressed p-AKT, p-GSK3β expression as well as β-catenin nuclear translocation, while inhibition of GSK3β activity or exogenous polyamines could restore SSAT-induced decreases in the protein expression of p-AKT, p-GSK3β and β-catenin. Conversely, knockdown of SSAT in Bel7402 hepatocellular carcinoma cells and HT-29 colorectal carcinoma cells which expressed high levels of SSAT endogenously significantly promoted the expression of p-AKT, p-GSK3β as well as β-catenin nuclear translocation. Taken together, our results indicated depletion of polyamines by SSAT significantly inhibited cell proliferation, migration and invasion through AKT/GSK3β/β-catenin signaling pathway in hepatocellular carcinoma and colorectal cancer cells.

Background and purpose: Metastasis is an important cause of death in breast cancer patients. Anti-metastatic agents are urgently needed since standard chemotherapeutics cannot diminish the metastatic rate. Actein, a cycloartane triterpenoid, has been demonstrated to exhibit anti-angiogenic and anti-cancer activities. Its anti-metastatic activity and underlying mechanisms were evaluated in the present study. Methods: The effects of actein on the proliferation, cell cycle phase distribution, migration, motility and adhesion were evaluated using two human breast cancer cell lines, MDA-MB-231 (estrogen receptor-negative) and MCF-7 cells (estrogen receptor-positive) in vitro. Western blots and real-time PCR were employed to examine the protein and mRNA expression of relevant signaling pathways. A human metastatic breast cancer cell xenograft model was established in transparent zebrafish embryos to examine the anti-migration effect of actein in vivo. Results: In vitro results showed that actein treatment significantly decreased cell proliferation, migration and motility. Furthermore, actein significantly caused G1 phase cell cycle arrest and suppressed the protein expression of matrix metalloproteinases of MDA-MB-231 cells. In addition, actein inhibited breast cancer cell adhesion to collagen, also reduced the expression of integrins. Actein treatment down-regulated the protein expression of epidermal growth factor receptor (EGFR), AKT and NF-κB signaling proteins. In vivo results demonstrated that actein (60 μM) significantly decreased the number of zebrafish embryos with migrated cells by 74% and reduced the number of migrated cells in embryos. Conclusion: Actein exhibited anti-proliferative, anti-adhesion and anti-migration activities, with the underlying mechanisms involved the EGFR/AKT and NF-kappaB signalings. These findings shed light for the development of actein as novel anti-migration natural compound for advanced breast cancer.

Abnormal activation of the RAF/MEK/ERK signaling pathway has been observed in breast cancer. Thus, a number of MEK inhibitors have been designed as one treatment option for breast cancer. Although some studies have found that these MEK inhibitors inhibit the growth of a variety of human cancer cells, some trials have shown that the use of MEK inhibitors as a treatment for breast cancer does not adequately improve survival for unknown reasons. In the present study, MEK inhibitor PD98059 was used to evaluate its anticancer effects on human breast cancer MCF-7 and MDA-MB-231 cells and to explore the possible mechanism of action. Our results revealed that MEK inhibitor PD98059 exhibited antiproliferative effects in a dose- and time-dependent manner in MCF-7 and MDA-MB-231 breast cancer cells. Conversely, incubation of MCF-7 and MDA-MB-231 cells with PD98059 promoted their migration. Further investigation disclosed that the enhanced ability of migration promoted by PD98059 was dependent on β-catenin nuclear translocation in the MCF-7 and MDA-MB‑231 cells. Subsequent experiments documented that activation of EGFR signaling induced by PD98059 increased the amount of β-catenin in the nucleus. Taken together, our findings may elucidate a possible mechanism explaining the ineffectiveness of MEK inhibitors in breast cancer treatment and improve our understanding of the role of MEK in cancer.

Actein is a triterpene glycoside isolated from the rhizomes of Cimicifuga foetida (Chinese herb “shengma”) which could inhibit the growth of breast cancer cells. Nevertheless, the effect of actein on angiogenesis, which is an essential step for tumor growth and metastasis, has never been reported. Hence, this study aimed to investigate the in vitro and in vivo effects of actein on angiogenesis using human microvascular endothelial cells (HMEC-1), matrigel plug and tumor-bearing mouse models. Our results showed that actein significantly inhibited the proliferation, reduced the migration and motility of endothelial cells, and it could suppress the protein expressions of VEGFR1, pJNK and pERK, suggesting that JNK/ERK pathways were involved. In vivo results showed that oral administration of actein at 10 mg/kg for 7 days inhibited blood vessel formation in the growth factor-containing matrigel plugs. Oral actein treatments (10–15 mg/kg) for 28 days resulted in decreasing mouse 4T1 breast tumor sizes and metastasis to lungs and livers. The apparent reduced angiogenic proteins (CD34 and Factor VIII) expressions and down-regulated metastasis-related VEGFR1 and CXCR4 gene expressions were observed in breast tumors. Our novel findings provide insights into the use of actein for development of anti-angiogenic agents for breast cancer.

Breast invasive micropapillary carcinoma (IMPC) is a rare subtype of breast cancer with a high potential of lymph node metastasis, aggressive clinical behavior, and poor disease-free or overall survival. Expression of leucine zipper putative tumor suppressor 1 (LZTS1) was frequently lost or reduced in breast cancer tissues. This study investigated the expression of LZTS1 protein in breast IMPC tissues using immunohistochemistry. In addition, somatic LZTS1 mutations and promoter methylation were assessed to determine an association with clinicopathological data from IMPC patients. LZTS1 protein was downregulated in 62 (62 %) of 100 IMPC tissue samples and was significantly associated with lymph node metastasis (P