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Collagen triple helix repeat containing 1 (CTHRC1) is a gene that has been associated with tumor progression in human prostate cancer (PC). The tumor immune microenvironment has been linked with disease outcome in PC. In the present study, the correlation between CTHRC1 with PC recurrence and the tumor immunological microenvironment was investigated. Using the data supplied by the Tumor Immune Estimation Resource (TIMER), the expression of CTHRC1, programmed cell death protein 1 (PD‑1), and programmed cell death 1 ligand 1 (PD‑L1) were analyzed. Immunohistochemical staining of CTHRC1, PD‑1 and PD‑L1 was performed using a tissue microarray construction of prostate adenocarcinoma (PRAD) specimens. In PRAD, an association was reported between the CTHRC1 expression and the disease free survival (DFS) rate (P=0.022). Overexpression of CTHRC1 was correlated with increased levels of PD‑1 (R=0.272, P=0.021) and PD‑L1 (R=0.298, P=0.016), elevated levels of infiltrating B cells (P=9.51e‑11), CD4+ cells (P=1.51e‑11), macrophages (P=8.25e‑5), neutrophils (P=2.17e‑9) and dendritic cells (P=3.13e‑13). Bioinformatics analysis revealed that CTHRC1 was correlated with the expression levels of matrix metalloproteinase‑9, mucin 1 and solute carrier organic anion transporter family member 2B1 genes, which exert an influence in PRAD. The occurrence of this condition is most likely to be associated with regulation of the tumor microenvironment. Taken together, we demonstrated that the prognosis and immunity of PC are closely linked to CTHRC1 upregulation. Furthermore, these results suggest that the immune function of PC may be suppressed by CTHRC1‑targeting therapy.

Background: Japanese scallop has been cultured on a large-scale in China for many years. However, serious marine pollution in recent years has resulted in considerable loss to this industry. Moreover, due to the lack of genomic resources, limited research has been carried out on this species. To facilitate the understanding at molecular level immune and stress response mechanism, an extensive transcriptomic profiling and digital gene expression (DGE) database of Japanese scallop upon cadmium exposure was carried out using the Illumina sequencing platform., Results: RNA-seq produced about 112 million sequencing reads from the tissues of adult Japanese scallops. These reads were assembled into 194,839 non-redundant sequences with open reading frame (ORF), of which 14,240 putative amino acid sequences were assigned biological function annotation and were annotated with gene ontology and eukaryotic orthologous group terms. In addition, we identified 720 genes involved in response to stimulus and 302 genes involved in immune-response pathways. Furthermore, we investigated the transcriptomic changes in the gill and digestive gland of Japanese scallops following cadmium exposure using a tag-based DGE system. A total of 7,556 and 3,002 differentially expressed genes were detected, respectively, and functionally annotated with KEGG pathway annotations., Conclusion: This study provides a comprehensive transcripts sequence resource for the Japanese scallop and presents a survey of gene expression in response to heavy metal exposure in a non-model marine invertebrate via the Illumina sequencing platform. These results may contribute to the in-depth elucidation of the molecular mechanisms involved in bivalve responses to marine pollutants.

Ovarian cancer (OC) is the seventh most prevalent type of cancer in women and the second most common cause of cancer-related deaths in women worldwide. Because of the high rates of relapse, there is an immediate and pressing need for the discovery of innovative sensitive biomarkers for OC patients. Using TCGA and GSE26712 datasets, we were able to identify 17 survival-related DEGs in OC that had differential expression. CLDN4 was the gene that caught our attention the most out of the 17 important genes since its expression was much higher in OC samples than in nontumor samples. The findings of the ROC assays then confirmed the diagnostic utility of the test in screening OC specimens to differentiate them from nontumor specimens. Patients with high CLDN4 expression predicted a shorter overall survival (OS) and disease-specific survival (DSS) than those with low CLDN4 expression, according to clinical research. Patients with low CLDN4 expression predicted longer OS and DSS. Analysis using both univariate and multivariate techniques revealed that CLDN4 expression was an independent factor associated with a poor prognosis for OS and DSS. Based on multivariate analysis, the C -indexes and calibration plots of the nomogram suggested an effective predictive performance for OC patients. After that, we investigated whether or not there was a link between the infiltration of immune cells and the expression of the CLDN4 gene. We found that the expression of CLDN4 was positively associated with Th17 cells, NK CD56bright cells, while negatively associated with Th2 cells, pDC, and T helper cells. In the end, we carried out RT-PCR on our cohort and confirmed that the level of CLDN4 expression was noticeably elevated in OC specimens in comparison to nontumor tissues. The diagnostic usefulness of CLDN4 expression for OC was also validated by the findings of ROC tests. Thus, our findings revealed that CLDN4 may serve as a predictive biomarker in OC to assess both the clinical outcome of OC patients and the level of immune infiltration. [ABSTRACT FROM AUTHOR]

Ginger (Zingiber officinale) is one of the most valued spice plants worldwide; it is prized for its culinary and folk medicinal applications and is therefore of high economic and cultural importance. Here, we present a haplotype-resolved, chromosome-scale assembly for diploid ginger anchored to 11 pseudochromosome pairs with a total length of 3.1 Gb. Remarkable structural variation was identified between haplotypes, and two inversions larger than 15 Mb on chromosome 4 may be associated with ginger infertility. We performed a comprehensive, spatiotemporal, genome-wide analysis of allelic expression patterns, revealing that most alleles are coordinately expressed. The alleles that exhibited the largest differences in expression showed closer proximity to transposable elements, greater coding sequence divergence, more relaxed selection pressure, and more transcription factor binding site differences. We also predicted the transcription factors potentially regulating 6-gingerol biosynthesis. Our allele-aware assembly provides a powerful platform for future functional genomics, molecular breeding, and genome editing in ginger. [ABSTRACT FROM AUTHOR]

Aim of the Study: Matrix metalloproteinases (MMPs) play a critical role in chronic obstructive pulmonary disease (COPD). This study investigated the role of mitogen-activated protein kinases (MAPKs) in MMP-9 secretion of BEAS-2B cells, a human bronchial epithelial cell line and U937 cells, a human myeloid leukaemia cell line, which could differentiate into macrophage, after LPS stimulation, and some details of involved signaling. Materials and Methods: MTT assay was used to measure cell viability. U937 cells were incubated for 48h with 100ng/ml PMA, and had a resting period of 24h with culture medium without PMA for differentiation of U937 cells into macrophages. For the experiments, U937 cells or BEAS-2B cells were pretreated with several inhibitors and then stimulated by LPS. Western blotting, quantitative real-time PCR, enzyme-linked immunosorbent assay (ELISA) and DNA binding activity assay were used for measuring the protein expression, RNA expression, cytokine production and DNA binding activity, respectively. Results: We found LPS induced MMP-9 expression and secretion were completely blocked by stress-activated protein kinase/jun kinase (SAPK/JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitors but not by p38 inhibitor. LPS-induced transactivation of AP-1 was also inhibited by JNK inhibitor SP600125 and ERK1/2 inhibitor PD98059. Conclusions: The present study suggests that in BEAS-2B cells and U937 cells, LPS probably activates ERK1/2 pathway and JNK pathway, which in turn initiate AP-1 activity, and leading to MMP-9 expression. Thus the ERK1/2 inhibitor and JNK inhibitor may have potential clinical value in treating COPD. [ABSTRACT FROM AUTHOR]

Abstract: The bivalve Mizuhopecten yessoensis has been greatly impacted by marine pollutants in northern China. To elucidate the toxicological mechanism of copper exposure on the immune system, we investigated differentially expressed genes (DEGs) and transcript abundance in M. yessoensis gill tissue using the deep-sequencing platform Illumina HiSeq™ 2000. In total, 1312 and 2237 genes were identified as significantly up- or down-regulated, respectively. In addition, significant enrichment analysis identified 9 GO terms and 38 pathways involved in the response to copper exposure. The analysis of immune-related transcripts revealed a complex repertoire of innate recognition receptors, including toll-like receptors, NOD-like receptors and RIG-like receptors. Downstream pathway effectors, such as apoptotic, lysosomal and C-type lectin transcripts, were also analyzed. These results will provide a resource for subsequent gene expression studies regarding heavy metal exposure and the identification of copper-sensitive biomarkers to monitor the aquaculture of M. yessoensis. [Copyright &y& Elsevier]

Parathyroid-hormone-related peptide (PTHrP) is an important regulator of chondrocyte differentiation in growth plates but little is known about its role in deer antler cartilage. The aim of the present study was to use the deer antler as a model to determine the possible role of PTHrP in regulating chondrocyte differentiation of deer antler. PTHrP and its receptor PTH1R mRNA were highly expressed in the perichondrium and cartilage of sika deer antler, as shown by in situ hybridization. Chondrocytes of deer antler were identified by toluidine blue staining of glycosaminoglycan and immunocytochemical staining of type II collagen (Col II). Treatment with PTHrP (1-34) reduced the expression of prehypertrophic chondrocyte marker Col IX and hypertrophic chondrocyte marker Col X. In order to confirm the mechanism of action of PTHrP, we initially examined the expression of cyclin D1, Bcl-2 and runt-related transcription factor 2 (Runx2) in sika deer antler by in situ hybridization and found that cyclin D1, Runx2 and Bcl-2 mRNA were also expressed in antler chondrocytes. Exogenous PTHrP induced the expression of cyclin D1 and Bcl-2 mRNA by various signalling pathways, whereas it inhibited Runx2 expression through PKA, p38MAPK, MEK and PI3K signalling pathways. Thus, PTHrP might promote the proliferation of antler chondrocytes and prevent their differentiation; it might furthermore influence the growth and development of sika deer antler. [ABSTRACT FROM AUTHOR]

Background: Japanese scallop has been cultured on a large-scale in China for many years. However, serious marine pollution in recent years has resulted in considerable loss to this industry. Moreover, due to the lack of genomic resources, limited research has been carried out on this species. To facilitate the understanding at molecular level immune and stress response mechanism, an extensive transcriptomic profiling and digital gene expression (DGE) database of Japanese scallop upon cadmium exposure was carried out using the Illumina sequencing platform. Results: RNA-seq produced about 112 million sequencing reads from the tissues of adult Japanese scallops. These reads were assembled into 194,839 non-redundant sequences with open reading frame (ORF), of which 14,240 putative amino acid sequences were assigned biological function annotation and were annotated with gene ontology and eukaryotic orthologous group terms. In addition, we identified 720 genes involved in response to stimulus and 302 genes involved in immune-response pathways. Furthermore, we investigated the transcriptomic changes in the gill and digestive gland of Japanese scallops following cadmium exposure using a tag-based DGE system. A total of 7,556 and 3,002 differentially expressed genes were detected, respectively, and functionally annotated with KEGG pathway annotations. Conclusion: This study provides a comprehensive transcripts sequence resource for the Japanese scallop and presents a survey of gene expression in response to heavy metal exposure in a non-model marine invertebrate via the Illumina sequencing platform. These results may contribute to the in-depth elucidation of the molecular mechanisms involved in bivalve responses to marine pollutants. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs (miRNAs) are small, non-coding 21–25 nt RNA molecules that play an important role in regulating gene expression. Little is known about the expression profiles and functions of miRNAs in skin and their role in pigmentation. Alpacas have more than 22 natural coat colors, more than any other fiber producing species. To better understand the role of miRNAs in control of coat color we performed a comprehensive analysis of miRNA expression profiles in skin of white versus brown alpacas. Results: Two small RNA libraries from white alpaca (WA) and brown alpaca (BA) skin were sequenced with the aid of Illumina sequencing technology. 272 and 267 conserved miRNAs were obtained from the WA and BA skin libraries, respectively. Of these conserved miRNAs, 35 and 13 were more abundant in WA and BA skin, respectively. The targets of these miRNAs were predicted and grouped based on Gene Ontology and KEGG pathway analysis. Many predicted target genes for these miRNAs are involved in the melanogenesis pathway controlling pigmentation. In addition to the conserved miRNAs, we also obtained 22 potentially novel miRNAs from the WA and BA skin libraries. Conclusion: This study represents the first comprehensive survey of miRNAs expressed in skin of animals of different coat colors by deep sequencing analysis. We discovered a collection of miRNAs that are differentially expressed in WA and BA skin. The results suggest important potential functions of miRNAs in coat color regulation. [ABSTRACT FROM AUTHOR]

Circular RNA (circRNA) is a noncoding RNA that can regulate a variety of biological processes. CircRNAs can regulate gene expression posttranscriptionally by acting as microRNA sponges. Many turtle species are remarkable organisms due to their reproductive processes. However, information on circRNA in the gonads of turtles is limited. In this study, 6, 121 circRNAs were identified in the testes and ovaries of Chinese soft-shelled turtles (Pelodiscus sinensis) using the Illumina platform, and 710 circRNAs were significantly differentially expressed (DE). The DE circRNAs included 541 upregulated and 169 downregulated circRNAs in the testes. GO and KEGG pathway analysis indicated that the DE circRNAs were enriched in several signaling pathways, including GnRH, Wnt, FoxO, Progesterone mediated oocyte maturation, and mTOR signaling pathways. Five DE circRNAs were randomly selected, and their relative expression levels in ovaries and testes were detected by quantitative real-time PCR. All of these circRNAs were differentially expressed. In addition, 9, 883 interactions between circRNAs and miRNAs were predicted in the turtles. Target genes of the miRNAs include a range of genes regulating gonadal development. Seven ceRNA networks (DE circRNAs-DE miRNAs-DE mRNAs), including 7 DE circRNAs, 11 DE miRNAs and 20 DE mRNAs, were constructed. The networks included Cdc6 , the miR-1 family, the miR-203 family, and the miR-302 family. The expression profile of gonadal circRNAs might help to elucidate the roles of nonprotein coding RNAs in turtle gonadal development. [Display omitted] • The targeted mRNAs of differentially expressed circRNAs in gonads are enriched in various GO terms and KEGG pathways. • CeRNA networks reveal the potential connection of circRNAs, miRNAs and mRNAs in gonads. • Some dysregulated circRNAs associate with gonadal development. [ABSTRACT FROM AUTHOR]

Abstract: Chronic neuropathic pain is a common symptom in clinical practice and patients with chronic pain are subject to a greatly impaired quality of life. Grafted genetically-modified cells secreting enkephalin have been considered an encouraging treatment for chronic pain. Importantly, the transplanted cell as a therapeutic agent should be reproducible, safe, and controllable. In this study, by combining a tetracycline-controlled (Tet-on) gene expression system and immortalized astrocytes, we attempted to engineer an immortalized astrocyte line carrying the human preproenkephalin gene (IASL/hPPE) under the transcriptional control of doxycycline. These cells were then implanted into the subarachnoid space of chronic constrictive injury (CCI) rats and their analgesic potential was investigated by behavioral tests. The results showed that the secretion of enkephalin from IASL/hPPE cells could be switched on and off under the regulation of doxycycline in a dose-dependent manner. In addition, the mechanical and thermal hyperalgesia induced by CCI was significantly alleviated during the 2–7week period after grafts of IASL/hPPE cells and the analgesic effect could be regulated by doxycycline. Moreover, spinal enkephalin level could be modulated by the presence or absence of doxycycline in drinking water. Taken together, these data suggest that regulatable release of enkephalin from transplanted cells near the spinal dorsal horn was able to reverse the development of chronic neuropathic pain. Although improvements in the Tet-on system are necessary, this may provide an alternative approach for ex vivo cell transplantation to treat chronic pain. [Copyright &y& Elsevier]

Simple Summary: Although adipocytes affect the metastatic behavior of cancer cells, the underlying molecular mechanisms remain largely elusive. Thereby, we sought to screen for the signaling pathways responsible for adipocyte-induced motility of breast cancer cells by employing a breast cancer cell/adipocyte coculture system. Our study revealed that adipocyte coculture stimulated PAI-1 expression in breast cancer cells to potentiate cell motility. Furthermore, we obtained evidence that adipocytes secreted leptin to activate OBR in breast cancer cells, which phosphorylated STAT3 to promote the transcription of PAI-1 and repress the expression of miR-34a as the negative regulator of PAI-1. Our study provides new evidence for the involvement of adipocytes in breast cancer evolution, which advances the evolving roles of stromal cells in tumor pathogenesis. The crosstalk between cancer cells and adipocytes is critical for breast cancer progression. However, the molecular mechanisms underlying these interactions have not been fully characterized. In the present study, plasminogen activator inhibitor-1 (PAI-1) was found to be a critical effector of the metastatic behavior of breast cancer cells upon adipocyte coculture. Loss-of-function studies indicated that silencing PAI-1 suppressed cancer cell migration. Furthermore, we found that PAI-1 was closely related to the epithelial-mesenchymal transition (EMT) process in breast cancer patients. A loss-of-function study and a mammary orthotopic implantation metastasis model showed that PAI-1 promoted breast cancer metastasis by affecting the EMT process. In addition, we revealed that leptin/OBR mediated the regulation of PAI-1 through the interactions between adipocytes and breast cancer cells. Mechanistically, we elucidated that leptin/OBR further activated STAT3 to promote PAI-1 expression via miR-34a–dependent and miR-34a–independent mechanisms in breast cancer cells. In conclusion, our study suggests that targeting PAI-1 and interfering with its upstream regulators may benefit breast cancer patients. [ABSTRACT FROM AUTHOR]

• Chromatin remodeler genes might be involved in gonadal differentiation of the turtle. • Cyp19a1 and Foxl2 exhibited female-specific expression at different embryonic stages. • Dmrt1 , Amh , and Sox9 displayed male-specific expression at different embryonic stages. • miRNA might play an essential role in early gonadal differentiation in Pelodiscus sinensis. The Chinese soft-shelled turtle (Pelodiscus sinensis) is extensively cultured in Asia for its nutritional and medical value. Gonadal differentiation is fantastic in turtles, whereas morphologic, mRNA, and miRNA expressions were insufficient in the turtle. In this study, ovaries and testes histomorphology analysis of 14–23 stage embryos were performed, and mRNA and miRNA expression profiles were analyzed. Histomorphology analysis revealed that gonads were undifferentiated at embryonic stage 14. Ovarian morphological differentiation became evident from stage 15, which was characterized by the development of the cortical region and degeneration of the medullary region. Concurrently, testicular morphological differentiation was apparent from stage 15, marked by the development of the medullary region and degeneration of the cortical region. qRT-PCR results showed that Cyp19a1 and Foxl2 exhibited female-specific expression at stage 15 and the expression increased throughout most of the embryonic development. Dmrt1 , Amh , and Sox9 displayed male-specific expression at stage 15 and tended to increase substantially at later developmental stages. The expression of miR-8356 and miR-3299 in ZZ gonads were significantly higher than that in ZW gonads at stage 15, 17 and 19, and they had the highest expression at stage 15. While the expression of miR-8085 and miR-7982 had the highest expression at stage 19. Furthermore, chromatin remodeler genes showed differential expression in female and male P. sinensis gonads. These results of master sex-differentiation genes and morphological characteristics would provide a reference for the research of sex differentiation and sex reversal in turtles. Additionally, the expression of chromatin remodeler genes indicated they might be involved in gonadal differentiation of P. sinensis. [ABSTRACT FROM AUTHOR]

6-pyruvoyl-tetrahydropterin synthase (PTPS) is the second key enzyme of the pteridine biosynthetic pathway and it plays vital roles in fish body color formation. In this study, Ccptps of koi carp (Cyprinus carpio L.) was cloned, identified and characterized. The full-length cDNA of Ccptps was 1140 bp and encodes for 139 amino acids. Multiple alignments revealed that the amino acids sequence of CcPTPS shared the highest identity to that of C. carpio , and Ccptps was clustered with cyprinid fishes in phylogenetic tree. Liver tissues of koi carp exhibited the highest expression of Ccptps , followed by muscle and skin tissues. During early developmental stages, the expression of Ccptps declined from 2 dph to 4 dph, and increased from 4 dph to 12 dph. The expressions of Ccptps in three color-related tissues (skin, scale and caudal fin) of whole red (WR) koi carp were significantly higher than that of whole while (WW) koi carp. Immunohistochemistry results of skin tissues showed that CcPTPS was mainly located in epidermis, stratum compactum of dermis and muscle layer, with the signal intensities in stratum compactum and muscle layer were stronger in WR koi carp compared to WW koi carp. Co-expressions of CcPTPS, CcSPR and CcXDH were detected in skin tissues of WW and WR koi carps, with CcPTPS exhibited stronger signal intensity compared to CcSPR and CcXDH. These findings imply that Ccptps is potentially involved in koi carp body color formation through the pteridine synthesis pathway. [Display omitted] • A pteridine synthesis related gene Ccptps was cloned and characterized in koi carp. • The expressions of Ccptps were closely related to tissue colors. • The co-expression of CcPTPS, CcSPR and CcXDH was detected in skin of koi carp. • Ccptps may be involved in body color formation through pteridine synthesis pathway. [ABSTRACT FROM AUTHOR]

The insulin-like growth factor 1 (IGF1) plays important roles in fish reproduction. To understand the role of IGF1 in steroidogenesis and oocyte maturation, we investigated expression pattern and functions of Igf1 during in vitro maturation of spotted steed (Hemibarbus maculatus) oocytes. Igf1 expression quantified by qPCR and western blotting revealed that both Igf1 mRNA and protein was significantly increased in mature follicles. The immunofluorescence analyses showed IGF1 protein signal was detected in the theca cells of ovarian follicles during vitellogenesis stage, and in both theca cells and granulosa cells of matured oocytes. Incubation of ovarian follicles with recombinant IGF1 (rIGF1) significantly enhanced the rate of spotted steed oocyte germinal vesicle breakdown (GVBD), a marker of oocyte maturation. qPCR analysis showed that the transcription of steroidogenesis-related genes (3β-hsd , 20β-hsd , Cyp17a and Cyp19a1a) were influenced after IGF1 treatment. In addition, the expression profiles of receptors of Igf1 , Fsh and Lh indicated that a cross talk response to IGF1 induced oocyte maturation. Furthermore, IGF1 treatment did not affect ERK protein expression but significantly upregulated CyclinB and AKT protein expression. All together, these findings help us to understand the molecular mechanism of IGF1 in regulation ovarian steroidogenesis and oocyte maturation of spotted steed. • Incubation of post vitellogenic follicles with rIGF1 promoted spotted steed oocyte maturation. • IGF1 treatment stimulated steroidogenesis-related genes, as well as gonadotropin receptors and Igf1r expression. • IGF1 induced oocyte maturation resulted from upregulation of CyclinB and AKT protein expression. [ABSTRACT FROM AUTHOR]

This study aims to investigate the effects of β-elemene on a mouse model of heart failure (HF) and to elucidate the underlying mechanisms in vitro approaches. In this study, left anterior descending (LAD)-induced HF mouse model and oxygen-glucose deprivation/recovery (OGD/R)-induced H9C2 model were leveraged to assess the therapeutic effects of β-elemene. Histological examination, western blot and quantitative real-time PCR analysis (RT-qPCR) and immunofluorescence staining was utilized to elucidate mechanism of β-elemene in lipid-induced inflammation. Results showed that β-elemene improved heart function in HF mice evidenced by the increase of cardiac ejection fraction (EF) and fractional shortening (FS) values. Furthermore, β-elemene administration rescued ventricular dilation, lipid accumulation, and inflammatory infiltration in arginal areas of mice myocardial infarction. At transcription level, β-elemene augmented the mRNA expression of fatty acid oxidation-associated genes, such as peroxisome proliferator-activated receptor-β (PPARβ). In vitro, treatment of β-elemene increased carnitine palmitoyltransferase 1A (CPT1A) and sirtuin 3 (SIRT3). Hallmarks of inflammation including the nuclear translocation of nuclear factor κB (NF-κB) and the degradation of inhibitory κBα (IκBα) were significantly suppressed. Consistently, we observed down-regulation of interleukin-6 (IL-6) and pro-inflammatory cytokines (such as TNFα) in β-elemene treated H9C2 cells. Finally, molecular docking model predicted an interaction between β-elemene and PPARβ protein. Furthermore, β-elemene increased the expression of PPARβ, which was validated by antagonist of PPARβ and siRNA for PPARβ. [ABSTRACT FROM AUTHOR]

Danqi Pill, composed of the root of Salvia miltiorrhiza Bunge and the root of Panax notoginseng , is effective in the clinical treatment of myocardial ischemia in coronary heart diseases. A number of studies have shown that autophagy plays an essential role in cardiac function and energy metabolism, and disordered autophagy is associated with the progression of heart failure. However, the effect and mechanism of Danqi pill on autophagy have not been reported yet. This study aims to elucidate whether Danqi pill restores autophagy to protect against HF and its potential mechanism. Left anterior descending ligation was established to induce an HF rat model, H 2 O 2 -stimulated H9C2 cells model was conducted to clarify the effects and potential mechanism of Danqi pill. In vivo, Danqi pill (1.5 g/kg) were orally administered for four weeks and Fenofibrate (10 mg/kg) was selected as a positive group. In vitro, Danqi pill (10–200 μg/mL) was pre-cultured for 24 h and co-cultured with H 2 O 2 stimulation for 4 h. Importantly, transmission electron microscopy and fluorescence GPF-mRFP-LC3 reporter system were combined to monitor autophagy flux. Furtherly, we utilized Compound C, a specific AMPK inhibitor, to validate whether the autophagy was mediated by AMPK-TSC2-mTOR pathway. Danqi pill significantly improved cardiac function and myocardial injury in HF rats. Intriguingly, Danqi pill potently regulated autophagy mainly by promoting the formation of autophagosomes in vivo. Further results demonstrated that expressions of p-AMPK (P < 0.001) and p-TSC2 (P < 0.001) in cardiac tissue were upregulated by Danqi pill, accompanied with downregulation of p-mTOR (P < 0.01) and p-ULK1(P < 0.01). In parallel with the vivo experiment, in vitro study indicated that Danqi pill dramatically restored autophagy flux and regulated expressions of critical autophagy-related molecules. Finally, utilization of Compound C abrogated the effects of Danqi pill on autophagy flux and the expressions of p-TSC2 (P < 0.05), p-mTOR (P < 0.01) and p-ULK1 (P < 0.05). Danqi pill could improve cardiac function and protect against cardiomyocytes injury by restoring autophagy via regulating the AMPK-TSC2-mTOR signaling pathway. Image 1 [ABSTRACT FROM AUTHOR]