Elucidation of the Mechanisms and Molecular Targets of Sanhuang Xiexin Decoction for Type 2 Diabetes Mellitus Based on Network Pharmacology.

Sanhuang Xiexin Decoction (SXD) is commonly used to treat type 2 diabetes mellitus (T2DM) in clinical practice of traditional Chinese medicine (TCM). In order to elucidate the specific analysis mechanisms of SXD for T2DM, the method of network pharmacology was applied to this article. First, the effective ingredients of SXD were obtained and their targets were identified based on the TCMSP database. The T2DM-related targets screened from the GEO database were also collected by comparing the differential expressed genes between T2DM patients and healthy individuals. Then, the common targets in SXD-treated T2DM were obtained by intersecting the putative targets of SXD and the differential expressed genes of T2DM. And the protein-protein interaction (PPI) network was established using the above common targets to screen key genes through protein interactions. Meanwhile, these common targets were used for GO and KEGG analyses to further elucidate how they exert antidiabetic effects. Finally, a gene pathway network was established to capture the core one in common targets enriched in the major pathways to further illustrate the role of specific genes. Based on the data obtained, a total of 67 active compounds and 906 targets of SXD were identified. Four thousand one hundred and seventy-six differentially expressed genes with a P value < 0.005 and ∣log2(fold change) | >0.5 were determined between T2DM patients and control groups. After further screening, thirty-seven common targets related to T2DM in SXD were finally identified. Through protein interactions, the top 5 genes (YWHAZ, HNRNPA1, HSPA8, HSP90AA1, and HSPA5) were identified. It was found that the functional annotations of target genes were associated with oxygen levels, protein kinase regulator, mitochondria, and so on. The top 20 pathways including the PI3K-Akt signaling pathway, cancers, HIF-1 signaling pathway, and JAK-STAT signaling pathway were significantly enriched. CDKN1A was shown to be the core gene in the gene-pathway network, and other several genes such as CCND1, ERBB2, RAF1, EGF, and VEGFA were the key genes for SXD against T2DM. Based on the network pharmacology approach, we identified key genes and pathways related to the prognosis and pathogenesis of T2DM and also provided a feasible method for further studying the chemical basis and pharmacology of SXD.

Prunin suppresses viral IRES activity and is a potential candidate for treating enterovirus A71 infection.

Human enterovirus A71 (HEVA71) causes hand, foot, and mouth disease (HFMD) in young children and is considered a major neurotropic pathogen but lacks effective antivirals. To identify potential therapeutic agents against HFMD, we screened a 502-compound flavonoid library for compounds targeting the HEVA71 internal ribosome entry site (IRES) that facilitates translation of the HEVA71 genome and is vital for the production of HEVA71 viral particles. We validated hits using cell viability and viral plaque assays and found that prunin was the most potent inhibitor of HEVA71. Downstream assays affirmed that prunin disrupted viral protein and RNA synthesis and acted as a narrow-spectrum antiviral against enteroviruses A and B, but not enterovirus C, rhinovirus A, herpes simplex 1, or chikungunya virus. Continuous HEVA71 passaging with prunin yielded HEVA71-resistant mutants with five mutations that mapped to the viral IRES. Knockdown studies showed that the mutations allowed HEVA71 to overcome treatment-induced suppression by differentially regulating recruitment of the IRES trans-acting factors Sam68 and hnRNPK without affecting the hnRNPA1-IRES interaction required for IRES translation. Furthermore, prunin effectively reduced HEVA71-associated clinical symptoms and mortality in HEVA71-infected BALB/c mice and suppressed hepatitis C virus at higher concentrations, suggesting a similar mechanism of prunin-mediated IRES inhibition for both viruses. These studies establish prunin as a candidate for further development as a HEVA71 therapeutic agent.

Hsp27 Responds to and Facilitates Enterovirus A71 Replication by Enhancing Viral Internal Ribosome Entry Site-Mediated Translation.

Enterovirus 71 (EV-A71) is a human pathogen that causes hand, foot, and mouth disease (HFMD) and fatal neurological diseases, and no effective treatment is available. Characterization of key host factors is important for understanding its pathogenesis and developing antiviral drugs. Here we report that Hsp27 is one of the most upregulated proteins in response to EV-A71 infection, as revealed by two-dimensional gel electrophoresis-based proteomics studies. Depletion of Hsp27 by small interfering RNA or CRISPR/Cas9-mediated knockout significantly inhibited viral replication, protein expression, and reproduction, while restoration of Hsp27 restored such virus activities. Furthermore, we show that Hsp27 plays a crucial role in regulating viral internal ribosome entry site (IRES) activities by two different mechanisms. Hsp27 markedly promoted 2Apro-mediated eukaryotic initiation factor 4G cleavage, an important process for selecting and initiating IRES-mediated translation. hnRNP A1 is a key IRES trans-acting factor (ITAF) for enhancing IRES-mediated translation. Surprisingly, knockout of Hsp27 differentially blocked hnRNP A1 but not FBP1 translocation from the nucleus to the cytoplasm and therefore abolished the hnRNP A1 interaction with IRES. Most importantly, the Hsp27 inhibitor 1,3,5-trihydroxy-13,13-dimethyl-2H-pyran [7,6-b] xanthone (TDP), a compound isolated from a traditional Chinese herb, significantly protected against cytopathic effects and inhibited EV-A71 infection. Collectively, our results demonstrate new functions of Hsp27 in facilitating virus infection and provide novel options for combating EV-A71 infection by targeting Hsp27.IMPORTANCE Outbreaks of infections with EV-A71, which causes hand, foot, and mouth disease, severe neurological disorders, and even death, have been repeatedly reported worldwide in recent decades and are a great public health problem for which no approved treatments are available. We show that Hsp27, a heat shock protein, supports EV-A71 infection in two distinct ways to promote viral IRES-dependent translation. A small-molecule Hsp27 inhibitor isolated from a traditional Chinese medicinal herb effectively reduces virus yields. Together, our findings demonstrate that Hsp27 plays an important role in EV-A71 infection and may serve as an antiviral target.

Antiviral activities of Schizonepeta tenuifolia Briq. against enterovirus 71 in vitro and in vivo.

No effective drug is currently available for treatment of enterovirus 71 (EV71) infection. Schizonepeta tenuifolia Briq. (ST) has been used as a herbal constituent of traditional Chinese medicine. We studied whether the aqueous extract of Schizonepeta tenuifolia Briq (STE) has antiviral activity. STE inhibited replication of EV71, as evident by its ability to diminish plaque formation and cytopathic effect induced by EV71, and to inhibit the synthesis of viral RNA and protein. Moreover, daily single-dose STE treatment significantly improved the survival of EV71-infected mice, and ameliorated the symptoms. Mechanistically, STE exerts multiple effects on enteroviral infection. Treatment with STE reduced viral attachment and entry; the cleavage of eukaryotic translation initiation factor 4 G (eIF4G) by EV71 protease, 2Apro; virus-induced reactive oxygen species (ROS) formation; and relocation of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) from the nucleus to the cytoplasm. It was accompanied by a decline in EV71-associated hyperphosphorylation of p38 kinase and EPS15. It is plausible that STE may inhibit ROS-induced p38 kinase activation, and subsequent hnRNP A1 relocation and EPS15-mediated membrane trafficking in infected cells. These findings suggest that STE possesses anti-EV71 activities, and may serve as health food or candidate antiviral drug for protection against EV71.

Lipid accumulation stimulates the cap-independent translation of SREBP-1a mRNA by promoting hnRNP A1 binding to its 5'-UTR in a cellular model of hepatic steatosis.

Non-alcoholic fatty liver disease (NAFLD) is a chronic disease characterized by accumulation of lipid droplets in hepatocytes. Enhanced release of non-esterified fatty acids from adipose tissue accounts for a remarkable fraction of accumulated lipids. However, the de novo lipogenesis (DNL) is also implicated in the etiology of the NAFLD. Sterol Regulatory Element-Binding Protein-1 (SREBP-1) is a transcription factor modulating the expression of several lipogenic enzymes. In the present study, in order to investigate the effect of lipid droplet accumulation on DNL, we used a cellular model of steatosis represented by HepG2 cells cultured in a medium supplemented with free oleic and palmitic fatty acids (FFAs). We report that FFA supplementation induces the expression of genes coding for enzymes involved in the DNL as well as for the transcription factor SREBP-1a. The SREBP-1a mRNA translation, dependent on an internal ribosome entry site (IRES), and the SREBP-1a proteolytic cleavage are activated by FFAs. Furthermore, FFA treatment enhances the expression and the nucleus-cytosolic shuttling of hnRNP A1, a trans-activating factor of SREBP-1a IRES. The binding of hnRNP A1 to the SREBP-1a IRES is also increased upon FFA supplementation. The relocation of hnRNP A1 and the consequent increase of SREBP-1a translation are dependent on the p38 MAPK signal pathway, which is activated by FFAs. By RNA interference approach, we demonstrate that hnRNP A1 is implicated in the FFA-induced expression of SREBP-1a and of its target genes as well as in the lipid accumulation in cells.

Heterogeneous Nuclear Ribonucleoprotein A1 Improves the Intestinal Injury by Regulating Apoptosis Through Trefoil Factor 2 in Mice with Anti-CD3-induced Enteritis.

BACKGROUND: The role of hnRNP A1 in the onset of intestinal inflammation remains unclear. This study investigated the function of hnRNP A1 in mice enteritis models. METHODS: C57Bl6/J mice were intraperitoneally injected with anti-CD3 antibodies to develop enteritis. In the DSS-induced colitis group, the mice were allowed free access to 3% DSS solution in their drinking water for 5 days. 3H-mannitol flux and complementary DNA array tests were used to assess the intestinal barrier function and messenger RNA (mRNA) expression, respectively. Real-time PCR was performed after immunoprecipitation with anti-hnRNP antibodies to determine the specific mRNA binding of hnRNP A1. RESULTS: The hnRNP A1 expression was increased in the intestine of the mouse at 24 hours after treatment with anti-CD3 antibodies and 5 days after starting DSS administration. Small interfering RNA (siRNA) against hnRNP A1 exacerbated the intestinal injuries in both models. According to the microarray analysis, trefoil factor 2 (TFF2) was identified as a candidate molecule targeted by hnRNP A1 in the anti-CD3 antibody-induced enteritis group. Moreover, the binding between hnRNP A1 and TFF2 mRNA significantly increased in the enteritis mice, and the administration of siRNA against either hnRNP A1 or TFF2 exacerbated the degree of intestinal injury. In the DSS-induced colitis group, treatment with the siRNA of hnRNP A1 worsened the intestinal injury, while the expression of TFF3 did not change. CONCLUSIONS: hnRNP A1 improves intestinal injury in anti-CD3 antibody-induced enteritis mice through the upregulation of TFF2, which regulates apoptosis and enhances epithelial restoration, whereas this molecule ameliorates DSS-induced colitis through a different pathway.

Chemical proteomics identifies heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as the molecular target of quercetin in its anti-cancer effects in PC-3 cells.

Quercetin, a flavonoid abundantly present in plants, is widely used as a phytotherapy in prostatitis and prostate cancer. Although quercetin has been reported to have a number of therapeutic effects, the cellular target(s) responsible for its anti-cancer action has not yet been clearly elucidated. Here, employing affinity chromatography and mass spectrometry, we identified heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) as a direct target of quercetin. A specific interaction between quercetin and hnRNPA1 was validated by immunoblotting and in vitro binding experiments. We found that quercetin bound the C-terminal region of hnRNPA1, impairing the ability of hnRNPA1 to shuttle between the nucleus and cytoplasm and ultimately resulting in its cytoplasmic retention. In addition, hnRNPA1 was recruited to stress granules after treatment of cells with quercetin for up to 48 h, and the levels of cIAP1 (cellular inhibitor of apoptosis), an internal ribosome entry site translation-dependent protein, were reduced by hnRNPA1 regulation. This is the first report that anti-cancer effects of quercetin are mediated, in part, by impairing functions of hnRNPA1, insights that were obtained using a chemical proteomics strategy.

The vitamin D response element-binding protein. A novel dominant-negative regulator of vitamin D-directed transactivation.

Vitamin D resistance in certain primate genera is associated with the constitutive overexpression of a non-vitamin D receptor (VDR)-related, vitamin D response element-binding protein (VDRE-BP) and squelching of vitamin d-directed transactivation. We used DNA affinity chromatography to purify proteins associated with non-VDR-VDRE binding activity from vitamin d-resistant New World primate cells. In electrophoretic mobility shift assays, these proteins bound specifically to either single-strand or double-strand oligonucleotides harboring the VDRE. Amino acid sequencing of tryptic peptides from a 34-kDa (VDRE-BP1) and 38-kDa species (VDRE-BP-2) possessed sequence homology with human heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and hnRNPA2, respectively. cDNAs bearing the open reading frame for both VDRE-BPs were cloned and used to transfect wild-type, hormone-responsive primate cells. Transient and stable overexpression of the VDRE-BP2 cDNA, but not the VDRE-BP1 cDNA, in wild-type cells with a VDRE-luciferase reporter resulted in significant reduction in reporter activity. These data suggest that the hnRNPA2-related VDRE-BP2 is a dominant-negative regulator of vitamin D action.

The protein Hrb57A of Drosophila melanogaster closely related to hnRNP K from vertebrates is present at sites active in transcription and coprecipitates with four RNA-binding proteins.

The hnRNP K protein is among the major hnRNA-binding proteins with a strong preference for cytidine-rich sequences. We have cloned a Drosophila hnRNP protein closely related to this vertebrate protein. The protein first identified by the monoclonal antibody Q18 is encoded by a gene located in 57A on polytene chromosomes and has been consequently named Hrb57A. The amino acid sequence of the Hrb57A KH domains and their overall organisation in the protein are remarkably similar to the vertebrate proteins. As the hnRNP K in vertebrates the M(r) 55 000 Drosophila Hrb57A/Q18 protein strongly binds to poly(C) in vitro and is ubiquitously present in nuclei active in transcription. On polytene chromosomes it is found in many puffs and minipuffs. Hrb57A/Q18 specifically coprecipitates four other proteins: Hrb87F/P11 a Drosophila hnRNP A1 homologue, the hnRNA-binding protein S5, the RNA recognition motif-containing protein NonA and the RNA-binding zinc finger-containing protein on ecdysone puffs PEP/X4.

Regulation of Ich-1 pre-mRNA alternative splicing and apoptosis by mammalian splicing factors.

The importance of alternative splicing in regulating apoptosis has been suggested by findings of functionally antagonistic proteins generated by alternative splicing of several genes involved in apoptosis. Among these, Ich-1 (also named as caspase-2) encodes a member of the caspase family of proteases. Two forms of Ich-1 are produced as a result of alternative splicing: Ich-1L, which causes apoptosis, and Ich-1S, which prevents apoptosis. The precise nature of Ich-1 alternative splicing and its regulation remain unknown. Here, we show that the production of Ich-1L and Ich-1S transcripts results from alternative exclusion or inclusion of a 61-bp exon. Several splicing factors can regulate Ich-1 splicing. Serine-arginine-rich proteins SC35 and ASF/SF2 promote exon skipping, decreasing the ratio of Ich-1S to Ich-1L transcripts; whereas heterogeneous nuclear ribonucleoprotein A1 facilitates exon inclusion, increasing this ratio. Furthermore, in cultured cells, SC35 overexpression increases apoptosis; whereas heterogeneous nuclear ribonucleoprotein A1 overexpression decreases apoptosis. These results provide the first direct evidence that splicing factors can regulate Ich-1 alternative splicing and suggest that alternative splicing may be an important regulatory mechanism for apoptosis.

Heterogeneous nuclear ribonucleoprotein A1 catalyzes RNA.RNA annealing.

Within the nucleus, pre-mRNA molecules are complexed with a set of proteins to form heterogeneous nuclear ribonucleoprotein complexes. A1, an abundant RNA binding protein present in these complexes, has been shown to bind selectively to single-stranded RNAs and destabilize base-pairing interactions. In this study.A1 is shown to promote the rate of annealing of complementary RNA strands greater than 300-fold under a wide range of salt concentration and temperature. Maximal annealing is observed under saturating or near saturating concentrations of protein, but annealing decreases sharply at both higher and lower concentrations of A1. Kinetic analysis shows that the rate of annealing is not strictly first or second order with respect to RNA at a ratio of protein/RNA that gives optimal rates of annealing. This result suggests that A1 protein may affect more than one step in the annealing reaction. Two polypeptides representing different domains of A1 were also examined for annealing activity. UP1, a proteolytic fragment that represents the N-terminal two-thirds of A1, displays very limited annealing activity. In contrast, a peptide consisting of 48 amino acid residues from the glycine-rich C-terminal region promotes annealing at a rate almost one-quarter that observed with intact A1. The RNA.RNA annealing activity of A1 may play a role in pre-mRNA splicing and other aspects of nuclear mRNA metabolism.