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Additional file 1. Supporting information.

Background Myocardial injury is a frequent complication after cardiac surgery with cardiopulmonary bypass (CPB). This study aimed to test the hypothesis that melatonin could attenuate myocardial injury in a rat CPB model. Methods Eighteen male Sprague-Dawley rats were randomly divided into three groups, n = 6 for each group: the sham operation (SO) group, CPB group and melatonin group. Rats in the SO group underwent cannulation without CPB, rats in CPB group intraperitoneal injected an equal volume of vehicle daily for 7 days before being subjected to CPB and rats in melatonin group intraperitoneal injected 20 mg/kg of melatonin solution daily for 7 days before being subjected to CPB. After 120 min for CPB, the expression levels of plasma interleukin (IL) -6, IL-1β, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), creatine kinase (CK) -MB and cardiac troponin T (cTnT) were measured. Reactive oxygen species (ROS) were detected by dihydroethidium (DHE). Apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. Mitochondrial damage and autophagosomes were detected by electron microscopy. Apoptosis inducing factor (AIF) was detected by immunofluorescence. The expression of B cell lymphoma/leukemia2 associated X (Bax), B cell lymphoma/leukemia 2 (Bcl-2), cytochrome C (Cyto-C), cleaved caspase-9, AKT, p-AKT, signal transducer and activator of transcription 3 (STAT3), p-STAT3, LC3, P62, mechanistic target of rapamycin kinase (mTOR), p-mTOR and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined using western blotting. Results Melatonin significantly decreased the levels of IL-1β, IL-6, MDA, CK-MB and cTnT and increased the levels of SOD and GSH-Px, all of which were altered by CPB. Melatonin reduced cardiomyocyte superoxide production, the apoptosis index and autophagy in cardiomyocytes induced by CPB. The AKT, STAT3 and mTOR signaling pathways were activated by melatonin during CPB. Conclusion Melatonin may serve as a cardioprotective factor in CPB by inhibiting oxidative damage, apoptosis and autophagy. The AKT, STAT3 and mTOR signaling pathways were involved in this process.

Background/Aims: Hepatocyte-like cells derived from human pluripotent stem cells could be an important cell source for hepatocyte transplantation. The present study investigated the effect of retaining mTeSR1 medium during hepatic differentiation on hepatocyte-like cells in vitro. Methods: Human embryonic stem cell line H1 were treated with activin A and bone morphogenetic protein 4 (BMP4) for definitive endoderm (DE) cell induction and subsequently treated with BMP2 and fibroblast growth factor 4 (FGF4) for early hepatic cell induction. Hepatocyte growth factor (HGF) and fibroblast growth factor (KGF) were added for early hepatic cell expansion and then mixed with oncostatin-M for maturation. During DE induction, 0%, 25%, 50% and 75% concentrations of mTeSR1 medium were separately added for early hepatic induction and expansion. For optimization, the expression levels of SRY-related HMG-box 17 (SOX17) and forkhead box A2 (FOXA2) at day 4, alpha fetoprotein (AFP) and hepatocyte nuclear factor 4α (HNF4α) at day 15, and albumin (ALB) at day 25 were quantified in differentiated cells by qRT-PCR. The ALB-positive cell proportion was measured by flow cytometry. Functional tests including ALB secretion and indocyanine green (ICG) angiography uptake and release by ELISA, urea production by urea assay kit, and glycogen storage ability by periodic acid Schif reaction (PAS) staining were performed in the differentiated cells. The induced pluripotent stem (iPS) cells were used to examine whether the optimized method was suitable for differentiating iPS cells. DE and hepatic markers were detected by immunostaining, and functional testing was performed as described above. Flow cytometry with an Annexin V-FITC apoptosis detection kit and fluorescence microscopy with Hoechst 33258 were used to analyze apoptosis in differentiated cells derived from H1 cells. Results: All differentiated cells with retention of 0%, 25%, 50% and 75% mTeSR1 expressed SOX17, FOXA2, AFP, HNF4α, and ALB, while higher expression levels were observed in differentiated cells in the 0% and 25% groups. The flow cytometry results showed that the proportion of ALB-positive differentiated cells derived from H1 cells was higher in the 25% mTeSR1 group than in other groups. However, no significant difference in ALB secretion, urea production, ICG uptake and release and glycogen storage ability was detected between the 25% and 0% groups. The iPS cells could differentiate into hepatocyte-like cells with 25% mTeSR1 retention. The apoptosis ratio of differentiated cells was lower in the 25% mTeSR1 group than in the 0% mTeSR1 group. Conclusion: Retaining 25% mTeSR1 medium during hepatic differentiation has been proposed to increase the percentage of ALB-positive cells and cell survival by decreasing cell apoptosis.

Background Atrial fibrillation (AF) is the most common sustained arrhythmia. DACT2 is a novel and important mediator of signaling pathways. The aim of this study was to investigate the clinical significance and functions of DACT2 expression in AF. Methods Immunohistochemistry was used to detect the DACT2 expression pattern in valvular disease patients. DACT2 was overexpressed in HL-1 cells and primary atrial fibroblasts. The expression levels of the potassium channel, the L-type calcium current channel, sodium ion channel proteins and collagen proteins were detected by real-time polymerase chain reaction (RT-PCR). The proteins involved in the Wnt and TGF-β signaling pathways were detected after DACT2 overexpression by western blotting. Results DACT2 expression was significantly associated with AF (P=0.016). The fibrosis ratio in the strong DACT2 expression group was significantly lower than that in the weak DACT2 expression group (weak: 0.198±0.091, strong: 0.129±0.064, P=0.048), and a negative correlation between DACT2 expression levels and fibrosis severity was observed (Spearman rho =-0.476, P=0.010). DACT2 significantly increased the expression levels of KCNE5 and decreased the levels of KCNH2 and SCN5A. Overexpression of DACT2 significantly inhibited the expression of collagen I and collagen III in primary rat atrial fibroblasts. DACT2 could facilitate β-catenin accumulation by reducing its phosphorylation at Thr41/Ser45 in HL-1 cells and inhibit the TGF-β signaling pathway in primary atrial fibroblasts. Conclusions DACT2 played a role in AF by regulating both structural and electrical atrial remodeling and by affecting β-catenin accumulation and TGF-β signaling, and it could serve as a protective factor against AF in valvular heart disease.

Background Immunity and inflammation are considered to be central features of pulmonary artery hypertension (PAH), in which macrophages are one of the main components of inflammatory cell infiltration around the pulmonary artery. M2b macrophages, which are different from M1 and M2 macrophages, are believed to have immunomodulatory activities and produce little fibrosis. The purpose of this study was to explore the effect of M2b macrophages on pulmonary artery smooth muscle cells (PASMCs) derived from monocrotaline-induced PAH rats. Methods PASMCs were cultured in serum-free medium, the supernatant of M0 macrophages, or the supernatant of M2b macrophages for 24 hours. Then cell proliferation was assessed by cell counting kit-8 and cell migration ability was detected by wound healing and transwell assays. The apoptosis rate of cells was determined by TUNEL staining and annexin V-PE/7-ADD staining. Western blot was used to detect the expression of Bcl-2 family proteins, cleaved caspase-9 and PI3K/Akt/FoxO3a pathway. LY294002 (a specific inhibitor of PI3K) was used to investigate its effect on PASMCs and its relationship with M2b macrophages. Results Conditioned medium from M2b macrophages significantly inhibited the proliferation and migration of PASMCs compared with the control group and M0 macrophage group. Furthermore, conditioned medium from M2b macrophages promote PASMC apoptosis and increased the expression of pro-apoptotic proteins Bax and cleaved caspase-9, inhibited the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl. Finally, conditioned medium from M2b macrophages inhibited the PI3K/Akt/FoxO3a pathway. Inhibition of PI3K/Akt/FoxO3a pathway also significantly inhibit the proliferation, migration, and apoptosis resistance of PASMCs. Conclusion Conditioned medium from M2b macrophages can inhibit the proliferation, migration, and apoptosis resistance of PASMCs, which may be at least partially by deregulating the PI3K/Akt/FoxO3a pathway.

Ethnopharmacological relevance Lung carcinoma (LC) is not only a kind of disease that seriously threatens human life but also an intractable problem in modern medicine. Jiao'e Mixture (JEM) is an innovative Chinese medicine formula with Chinese patent, which is composed of two herbal extracts with a specific ratio–zedoary turmeric oil and medicinal Zanthoxylum bungeanum Maxim(Z. bungeanum Maxim) seeds oil (ZMSO). Zedoary turmeric oil is extracted from dried rhizomes of Curcuma wenyujin Y.H.Chen et C. Ling, which has been reported have an anti-cancer effects. Medicinal ZMSO is a by-product of Z. bungeanum Maxim, refined from kernel shell separation, modern cold soaking and refining technology; JEM is used to treat Lung carcinoma (LC) patients in folk for many years. However, its therapeutic mechanisms for treating LC have not been fully explored. Aim of the study The purpose of this study was to explore the therapeutic mechanisms of JEM for treating LC. Materials and methods The action mechanism of JEM in LC treatment was analysed by comprehensive network pharmacology approach combined with experimental validation (in vivo and in vitro). Results Seventeen active compounds and 457 related targets were collected from the HERB, TCMSP, and Swiss Target Prediction platforms. Nine hundred and thirty-eight LC related targets were obtained from Gene Cards and OMIM databases. Finally, 140 overlapping targets were obtained, which representing the target of JEM in LC treatment. The pathway analysis showed that PI3K-AKT could be a potential pathway for JEM in LC treatment. In vivo results presented that JEM had a good effect in inhibiting the growth of LC tumour cells with high efficacy and low toxicity. In vitro experiments validated that JEM had inhibited LC cells’ proliferation, migration and invasion, and had induced cell apoptosis mainly via PI3K/Akt signalling pathways. Conclusion The anti-LC activity of JEM might via regulating the PI3K-AKT signalling pathways.This study may provide further evidence for the potential use of JEM in LC treatment.