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Background. Many studies have demonstrated the promising utility of DNA methylation and miRNA as biomarkers for colorectal cancer (CRC) early detection. However, mRNA is rarely reported. This study aimed to identify novel fecal-based mRNA signatures. Methods. The differentially expressed genes (DEGs) were first determined between CRCs and matched normal samples by integrating multiple datasets. Then, Least Absolute Shrinkage and Selection Operator (LASSO) regression was used to reduce the number of candidates of aberrantly expressed genes. Next, the potential functions were investigated for the candidate signatures and their ability to detect CRC and pan-cancers was comprehensively evaluated. Results. We identified 1841 common DEGs in two independent datasets. Functional enrichment analysis revealed they were mainly related to extracellular structure, biosynthesis, and cell adhesion. The CRC classifier was established based on six genes screened by LASSO regression. Sensitivity, specificity, and area under the ROC curve (AUC) for CRC detection were 79.30%, 80.40%, and 0.85 (0.76–0.92) in the training set, and these indexes achieved 93.20%, 41.80%, and 0.73 (0.65–0.83) in the testing set. For validation set, the sensitivity, specificity, and AUC were 98.90%, 98.00%, and 0.97 (0.94–0.99). The average sensitivities exceeded 90.00% for CRCs with different clinical features. For adenomas detection, the sensitivity and specificity were 74.50% and 64.00%. Besides, the six genes obtained an average AUC of 0.855 for pan-cancer detection. Conclusion. The six-gene signatures showed ability to detect CRC and pan-cancer samples, which could be served as potential diagnostic markers.

Ying Ning,* Yanju Gong,* Tianyan Zheng, Ya Xie, Shiqing Yuan, Weijun Ding School of Basic Medical Sciences, Chengdu University of Traditional Chinese Medicine, Chengdu, 611137, People’s Republic of China*These authors contributed equally to this workCorrespondence: Weijun Ding; Shiqing Yuan, Department of Fundamental Medicine, Chengdu University of Traditional Chinese Medicine, 1166 Liutai Road, Chengdu, 611137, People’s Republic of China, Tel + 86-28-61800219, Fax + 86-28-61800225, Email Dingweijun@cdutcm.edu.cn; yuanshiqing@cdutcm.edu.cnBackground: The increasing incidence of obesity and its complications has become a global public health problem. Lingguizhugan decoction (LGZGD) is a representative compound of traditional Chinese medicine (TCM) for metabolic diseases, such as nonalcoholic fatty liver disease, but its role in insulin resistance (IR) treatment is still less known. This study aims to evaluate the therapeutic properties of LGZGD on obesity-induced IR and explore the potential mechanism of LGZGD on gut microbiota and its metabolites in the treatment of IR.Methods: In this study, we induced an IR model in the form of high-fat diet (HFD) rats gavaged with LGZGD (1.64 g/kg BW) for three weeks. The IR status was measured by biochemical assays and oral glucose tolerance tests. The degrees of damage to liver function and the intestinal barrier were observed by hematoxylin and eosin (H&E) staining and immunohistochemistry. Alterations in intestinal microbiota and metabolites were assessed by 16S rRNA and an untargeted metabolomics platform.Results: Our results showed that after LGZGD treatment, the body weight, plasma insulin concentration and blood lipids were significantly decreased, and glucose tolerance and hepatic steatosis were ameliorated. In addition, small intestinal villi were restored, and the expression of Occludin was upregulated. The relative abundance of Akkermansia, Faecalibacterium and Phascolarctobacterium in the HFD-LGZG group was upregulated. Obesity-related metabolic pathways, such as bile secretion, biosynthesis of amino acids, phenylalanine metabolism, serotonergic synapse, protein digestion and absorption, taurine and hypotaurine metabolism, and primary bile acid biosynthesis, were changed. After LGZGD intervention, metabolites developed toward the healthy control group. In addition, the expression of bile acid metabolism related genes was also regulated in IR rats.Conclusion: We showed that LGZGD relieved IR, possibly by regulating the composition of the fecal microbiota and its metabolites. The above studies provide a basis for further study of LGZGD in the treatment of IR and its clinical application.Keywords: insulin resistance, IR, lingguizhugan decoction, LGZGD, intestinal microbiota, nontargeted metabolomics, high-fat diet, HFD

Hepatocellular carcinoma (HCC) is a major threat to human health due to its high lethality. Our previous studies suggested that Fuzheng Xiaozheng prescription (FZXZP), an effective Chinese medicine, demonstrated significant suppressive effects on HCC. However, its underlying mechanism remains largely unclear.This study aimed to investigate the anti-HCC mechanisms of FZXZP from transcriptomic sequencing based on a holistic perspective.Rat HCC model was induced by diethylnitrosamine, and then the model was administered with two doses of FZXZP, high and low. Sodium demethylcantharidate was used as a positive control. Subsequently, microarrays of circRNA, miRNA and mRNA were performed on the blank, model, high and low dose groups, respectively, and the competitive binding mechanisms among them were further analyzed by bioinformatics. Then, the circRNA-miRNA-mRNA networks were constructed to mine the targeted-RNAs of FZXZP in HCC, as well as to explore their potential regulatory mechanisms. Finally, functions and pathways of the FZXZP targeted genes in rat HCC were annotated with GO and KEGG, and qRT-PCR was performed to validate the accuracy of the above analyses in this study.The results showed that FZXZP significantly inhibited the development and progression of HCC in rats, improved the pathological conditions and suppressed the proliferation of HCC cells. Subsequently, after a series of screening, the competing endogenous RNA networks (circRNA-miRNA-mRNA), consisting of 2 circRNAs, 7 miRNAs and 104 mRNAs, were finally established. KEGG and GO analyses of the networks revealed that lipid metabolism related pathways, such as fatty acid metabolism, bile secretion and PPAR pathway, were significantly enriched. In the further hubgene network analysis, in addition to lipid metabolism, aberrant glucose metabolism was found to be ameliorated by G6pc and Pklr in hubgenes. Finally, the qRT-PCR analyses confirmed that the expression tendencies of the above targeted genes were correct and believable in transcriptomic sequencings, and qRT-PCR results of the genes closely related to proliferation, invasion and apoptosis of HCC also indicated the inhibitory effects of FZXZP on HCC obviously.FZXZP demonstrated significant anti-HCC effects through improving lipid and glucose metabolism, restoring the metabolic homeostasis of the liver via circRNA-miRNA-mRNA networks.

Fuzheng Xiaozheng prescription (FZXZP), a traditional Chinese medicine, which was derived from the famous decoction, Sanjiasan, in the book of "Wenyilun" in Ming dynasty. Due to its function of invigorating the circulation of blood in Chinese medicine, it was usually used for treating the liver cirrhosis, hepatocellular carcinoma (HCC), etc. Clinical application found that FZXZP exhibited satisfactory therapeutic effects in HCC treatments. However, we still know little about the underlying mechanisms.In this study, we aim to gain a deeper insight into the inhibiting effects of FZXZP on HCC rats and preliminarily elucidate the underlying intervention effects.Two doses of FZXZP were adopted to evaluate the therapeutic effects on rat HCC, and then the intervention effects were evaluated from different aspects. High performance liquid chromatography (HPLC) was used for the active compounds prediction in FZXZP. Finally, the mRNA-Seq was conducted to reveal the intervention mechanisms and the mechanisms were further validated by quantitative Real-time PCR (qRT-PCR) and lipid contents analyses.The results showed that FZXZP significantly alleviated the serum biochemical indicators and improved the pathological characteristics of HCC rats. Mechanistically, FZXZP could regulate some lipid related metabolisms, including arachidonic acid, linoleic acid and retinol, as well as improving the steroid hormone biosynthesis, to improve the inflammatory statuses and restoring ability of HCC livers, and these were further confirmed by our following analyses on serum lipid contents and cytokine expressions. In addition, FZXZP could also negatively regulate four extracellular growth factors which could result in the blocking of two cancer-related signaling pathways, Ras/MAPK and Ras/PI3K-Akt.Our results suggested that FZXZP demonstrated significant inhibiting effects on rat HCC progresses, and these may be realized by improving the inflammatory statuses and blocking the Ras/MAPK and Ras/PI3K-Akt signaling pathways.

Obesity is currently one of the most important challenges to public health worldwide. Acupuncture has been widely used to treat obesity. However, whether acupuncture regulates intestinal innate immunity via intestinal microbiota against obesity remains to be elucidated. In this study, electroacupuncture (EA) effectively reduced body weight and fat accumulation in obese mice persistently fed a high-fat diet. Full-length 16S rDNA sequencing showed dysbiotic microbiota in the cecum of obese mice. The composition and function of the cecal microbiota of obese mice were markedly restored after EA treatment. After 21 d of EA intervention, the expression of defensin alpha 5 (Defa5) was restored to healthy controls, whereas fat digestion and absorption genes including fabp1 were markedly decreased in the jejunum of obese mice. The Defa5 levels were positively correlated with the family Lachnospiraceae and negatively correlated with obesity indexes. EA also reduced tissue inflammation, ameliorated misaligned glucose tolerance, and inhibited key genes for intestinal lipid absorption. In summary, EA exerted an anti-obesity effect by promoting intestinal defensins, rescuing dysbiotic cecal microbiota, and reducing lipid absorption in a synergistic mode. We present for the first time the key role of alpha defensins in the relationship between gut microbiota and disease during electroacupuncture treatment of obesity. The mucosal innate immunity seems to have a stronger ability to shape the microbiota than dietary factors.

Bufonis (VB), an animal drug called Chansu in China, is the product of the secretion of Bufo gargarizans Cantor or B. melanostictus Schneider. As a traditional Chinese medicine (TCM) for a long time, it has been widely used in the treatment of heart failure, ulcer, pain, and various cancers. Cinobufaginn (CNB), the cardiotonic steroid or bufalene lactone extracted from VB, has the effects of detoxification, detumescence, and analgesia.The present study aimed to define the effects of CNB on non-small-cell lung cancer (NSCLC) and identify the potential molecular mechanisms.A549 cells were treated with cinobufagin and cell viability, apoptosis, migration, and invasion were then evaluated using Cell Counting Kit-8 (CCK8) assays, flow cytometry, and Transwell assays, respectively. Moreover, the levels of proliferating cell nuclear antigen (PCNA), cytokeratin8 (CK8), poly ADP-ribose polymerase (PARP), Caspase3, Caspase8, B-cell lymphoma/lewkmia-2(Bcl-2), Bcl2-Associated X(Bax), forkhead box O1 (FOXO1), and euchromatic histone-lysine N-methyltransferase2 (G9a, EHMT2) in A549 cells were evaluated using qRT-PCR and/or Western blot analysis (WB), Co-IP, immunofluorescence, and immunohistochemistry. An in vivo imaging system, TUNEL, Immunofluorescence, and immunohistochemistry were also used to detect proliferating cell nuclear antigen(PCNA), Ki67, E-Cadherin(E-Cad), FOXO1, and G9a in mouse xenograft model experiments.CNB suppressed cell proliferation, migration, and invasion but promoted apoptosis in A549 cells in a dose- and time-dependent manner, while cinobufagin had no cytotoxic effect on BEAS-2B cells. In vivo, cinobufagin inhibited the proliferation, migration, and invasion of A549 cells and promoted their apoptosis. The occurrence of the above phenomena was accompanied by an increase in FOXO1 expression and a decrease in G9a expression. In A549 cells, CNB did not reverse the changes in the proliferation, migration, invasion, and apoptosis of A549 cells after FOXO1 was successfully silenced.Our study provides the first evidence that cinobufagin suppresses the malignant biological behaviours of NSCLC cells in vivo and in vitro and suggests that mechanistically, this effect may be achieved by inhibiting the expression of the histone methyltransferase G9a and activating the tumour suppressor gene FOXO1. Taken together, our findings provide important insights into the molecular mechanism underlying cinobufagin's anticancer activity, and suggest that cinobufagin could be a candidate for targeted cancer therapy.

Ethnopharmacological relevance Fuzheng Xiaozheng prescription (FZXZP) is a traditional Chinese medicine (TCM) that was derived from Sanjiasan, a famous decoction documented in the book of Wenyilun in Ming dynasty. Based on our years' clinic application, FZXZP demonstrated satisfactory therapeutic effects in cirrhosis and hepatocellular carcinoma (HCC) treatments. However, the underlying mechanisms are still largely unknown. Aim of study In this study, we aim to systematically evaluate the intervention effects of FZXZP on rat HCC and deeply elucidate the underlying regulative mechanisms on rat HCC. Materials and methods The HCC rats were induced by using diethylnitrosamine (DEN) and two doses of FZXZP were adopted to treat the HCC rats. Liver phenotype, blood chemistry and liver histopathology were used to evaluate the intervention effects. High performance liquid chromatography (HPLC) was conducted to analyze the components of FZXZP. Finally, miRNA-Seq and mRNA-Seq were performed to investigate the regulative mechanisms of FZXZP on rat HCC and qRT-PCR was carried out to verify the accuracies of the two RNA-Seqs. Results Results of liver phenotypes, blood chemistry and liver histopathology demonstrated that FZXZP significantly alleviated the liver damage, inhibited the progresses of HCC. Nine potential components were identified from FZXZP, and anti-cancer prediction suggested that almost all of them were reported to show an anti-cancer effect. Mechanistically, FZXZP was found to promote the lipid related metabolisms, improve the anti-inflammation ability by activating PPAR signaling pathway, arachidonic acid metabolism, bile secretion, etc. CONCLUSION: our results suggested that FZXZP significantly alleviated the rat HCC, mechanistically by improving the anti-inflammation ability and promoting the lipid related metabolisms.

Tetramethylpyrazine (TMP), one of the active ingredients of the Chinese herb Lingusticum Wallichii (Chuan Xiong) has been proved to protect the medulla oblongata from chronic hypoxia injury. However, the underlying mechanism remains unclear. The purpose of this study was to determine whether the protective effects of TMP are associated with the heme oxygenase-1 (HO-1) dependant pathway in adult rats. The morphological changes of neurons in the hypoglossal nucleus (12N), the nucleus ambiguus (Amb), the nucleus tractus solitarius (NTS), and the pre-Botzinger complex (pre-BotC) were investigated by Nissl staining; the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were measured to evaluate the anti-oxidant effect; some apoptosis parameters, Bax mRNA and Bcl-2 mRNA, were tested; and the double immunochemistry staining of active caspase-3/NeuN was performed. Meanwhile, the HO-1 protein expression and heme oxygenase (HO) activity were examined. Tin-protoporphyrin (SnPP), a potent inhibitor of HO, was used to further confirm the effect of HO-1. We found that TMP ameliorated the neuron loss in 12N, Amb and NTS, the decrease in SOD activity and the increase in MDA content, the decrease in Bcl-2 mRNA of medulla oblongata (P