Epicatechin gallate prevents the de novo synthesis of fatty acid and the migration of prostate cancer cells.

Lipid metabolism disorder caused by the upregulation of lipogenic genes is a typical feature of prostate cancer. The synthesis of fatty acids is enhanced to accelerate the development of prostate cancer and is considered as a potential therapeutic target. Epicatechin gallate, an active compound of green tea, has been reported to modulate lipid metabolism. In this research, the potential role of epicatechin gallate in prostate cancer cells was evaluated. The results indicated that epicatechin gallate downregulates the expression of acetyl-CoA carboxylase, ATP citrate lyase, and fatty acid synthase in prostate cancer cells and prostate xenograft tissues, suggesting that epicatechin gallate can inhibit de novo fatty acid synthesis. Moreover, epicatechin gallate significantly restrains the migration rather than the viability of prostate cancer cells. PI3K/AKT/mTOR signaling pathway, which exhibits regulatory effect on lipogenesis, is also inhibited under epicatechin gallate treatment, while pretreatment with AKT activator SC79 or mTOR activator MHY1485 blocks the inhibitory effect of epicatechin gallate on the expression of lipogenic genes and the migration of prostate cancer cells. In conclusion, this study revealed that epicatechin gallate impairs the synthesis of fatty acids via inhibition PI3K/AKT/mTOR signaling pathway and then attenuates the migration of prostate cancer cells.

[Effect of nuciferine on gut microbiota and inflammatory response in obese model mice].

The aim of this study was to elucidate the mechanism of nuciferine on alleviating obesity based on modulating gut microbiota, ameliorating chronic inflammation, and improving gut permeability. In this study, the obese model mice were induced by high-fat diet and then randomly divided into model group, and nuciferine group; some other mice of the same week age were fed with normal diet as normal group. In the modeling process, the mice were administered intragastrically(ig) for 12 weeks. In the course of both modeling and treatment, the body weight and food intake of mice in each group were measured weekly. After modeling and treatment, the Lee's index, weight percentage of inguinal subcutaneous fat, and the level of blood lipid in each group were measured. The pathological changes of adipocytes were observed by HE staining to evaluate the efficacy of nuciferine treatment in obese model mice. 16 S rRNA sequencing analysis was conducted to study the changes in diversity and abundance of gut microbiota after nuciferine treatment. Enzyme-linked immunosorbent assay(ELISA) and quantitative Real-time polymerase chain reaction(qPCR) were used to detect the levels of inflammatory factors interleukin-6(IL-6), interleukin-1ß(IL-1ß), tumor necrosis factor-α(TNF-α) and the expression of related genes in adipose tissue of mice in each group, so as to evaluate the effect of nuciferine on chronic inflammation of mice in obese model group. qPCR was used to detect the expression of occludin and tight junction protein 1(ZO-1)gene in colon tissure, so as to evaluate the effect of nuciferine on intestinal permeability of mice in obese group. Nuciferine decreased the body weight of obese mice, Lee's index, weight percentage of inguinal subcutaneous fat(P<0.05), and reduced the volume of adipocytes, decreased the level of total cholesterol(TC), triglyceride(TG), and low density lipoprotein cholesterol(LDL-C)(P<0.05) in serum, improved dysbacteriosis, increased the relative abundance of Alloprevotella, Turicibacter, and Lactobacillus, lowered the relative abundance of Helicobac-ter, decreased the expression of inflammatory cytokines IL-6, IL-1ß, and TNF-α genes in adipose tissue(P<0.01), decreased the levels of inflammatory cytokines IL-6, IL-1ß, and TNF-α in serum(P<0.05), and increased the expression of occludin and ZO-1 genes related to tight junction in colon tissue(P<0.01). Nuciferine could treat obesity through modulating gut microbiota, decreasing gut permeability and ameliorating inflammation.

Inhibition effect of glycyrrhiza polysaccharide (GCP) on tumor growth through regulation of the gut microbiota composition.

Glycyrrhiza Uralensis Polysaccharide (GCP), as a macromolecular polysaccharide extracted from the Traditional Chinese Medicine (TCM) - Licorice has been proved to inhibit tumor growth in vitro and in vivo; however, the specific anti-tumor mechanism of GCP needs to be further investigated. In this study, we explore the anti-tumor mechanism of GCP from the angle of gut microbiota. Colon carcinoma cells (CT-26) were used to set up a tumor-bearing mouse model. After 14 days of GCP treatment, the weights of tumors were significantly reduced. In addition, HE staining of tissue sections reflected that GCP could effectively inhibit tumor metastasis. 16SrRNA high-throughput sequencing of fecal samples showed a significant change between the model group and GCP group in the composition of gut microbiota. Subsequently, gut microbiota depletion and fecal transplantation experiments further confirmed the relationship between the anti-tumor effects of GCP and gut microbiota. Following depletion of gut microbiota, GCP cannot inhibit tumor growth. Fecal transplantation experiments found that transplanting the feces of GCP-treated mice, to a certain extent, could inhibit tumor growth and metastasis. These results indicate that Glycyrrhiza Polysaccharides exert anti-tumor effects by affecting gut microbiota composition.

Intra-domain Cross-talk Regulates Serine-arginine Protein Kinase 1-dependent Phosphorylation and Splicing Function of Transformer 2ß1.

Transformer 2ß1 (Tra2ß1) is a splicing effector protein composed of a core RNA recognition motif flanked by two arginine-serine-rich (RS) domains, RS1 and RS2. Although Tra2ß1-dependent splicing is regulated by phosphorylation, very little is known about how protein kinases phosphorylate these two RS domains. We now show that the serine-arginine protein kinase-1 (SRPK1) is a regulator of Tra2ß1 and promotes exon inclusion in the survival motor neuron gene 2 (SMN2). To understand how SRPK1 phosphorylates this splicing factor, we performed mass spectrometric and kinetic experiments. We found that SRPK1 specifically phosphorylates 21 serines in RS1, a process facilitated by a docking groove in the kinase domain. Although SRPK1 readily phosphorylates RS2 in a splice variant lacking the N-terminal RS domain (Tra2ß3), RS1 blocks phosphorylation of these serines in the full-length Tra2ß1. Thus, RS2 serves two new functions. First, RS2 positively regulates binding of the central RNA recognition motif to an exonic splicing enhancer sequence, a phenomenon reversed by SRPK1 phosphorylation on RS1. Second, RS2 enhances ligand exchange in the SRPK1 active site allowing highly efficient Tra2ß1 phosphorylation. These studies demonstrate that SRPK1 is a regulator of Tra2ß1 splicing function and that the individual RS domains engage in considerable cross-talk, assuming novel functions with regard to RNA binding, splicing, and SRPK1 catalysis.

Synthesis of a norcantharidin-tethered guanosine: Protein phosphatase-1 inhibitors that change alternative splicing.

Phosphorylation and dephosphorylation of splicing factors play a key role in pre-mRNA splicing events, and cantharidin and norcantharidin analogs inhibit protein phosphatase-1 (PP1) and change alternative pre-mRNA splicing. Targeted inhibitors capable of selectively inhibiting PP-1 could promote exon 7 inclusion in the survival-of-motorneuron-2 gene (SMN2) and shift the proportion of SMN2 protein from a dysfunctional to a functional form. As a prelude to the development of norcantharidin-tethered oligonucleotide inhibitors, the synthesis a norcantharidin-tethered guanosine was developed in which a suitable tether prevented the undesired cyclization of norcantharidin monoamides to imides and possessed a secondary amine terminus suited to the synthesis of oligonucleotides analogs. Application of this methodology led to the synthesis of a diastereomeric mixture of norcantharidin-tethered guanosines, namely bisammonium (1R,2S,3R,4S)- and (1S,2R,3S,4R)-3-((4-(2-(((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2-(hydroxymethyl)-4-methoxytetrahydrofuran-3-yl)oxy)oxidophosphoryl)oxy)ethyl)-phenethyl)(methyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylate, which showed activity in an assay for SMN2 pre-mRNA splicing.

The 5' untranslated region of the serotonin receptor 2C pre-mRNA generates miRNAs and is expressed in non-neuronal cells.

The serotonin receptor 2C (HTR2C) gene encodes a G protein-coupled receptor that is exclusively expressed in neurons. Here, we report that the 5' untranslated region of the receptor pre-mRNA as well as its hosted miRNAs is widely expressed in non-neuronal cell lines. Alternative splicing of HTR2C is regulated by MBII-52. MBII-52 and the neighboring MBII-85 cluster are absent in people with Prader-Willi syndrome, which likely causes the disease. We show that MBII-52 and MBII-85 increase expression of the HTR2C 5' UTR and influence expression of the hosted miRNAs. The data indicate that the transcriptional unit expressing HTR2C is more complex than previously recognized and likely deregulated in Prader-Willi syndrome.

Integrated 16S rRNA Sequencing and Untargeted Metabolomics Analysis to Reveal the Protective Mechanisms of Polygonatum sibiricum Polysaccharide on Type 2 Diabetes Mellitus Model Rats.

BACKGROUND: Polygonatum sibiricum polysaccharide (PSP) can improve insulin resistance and inhibit oxidative stress. However, the detailed anti-diabetic mechanism of PSP is still poorly defined. METHODS: In this study, the anti-diabetic, anti-inflammatory and anti-oxidative effects of PSP were evaluated on a type 2 diabetes mellitus (T2DM) rat model. Furthermore, we investigated the changes in gut microbiota and serum metabolites in T2DM rats after PSP treatment through 16S rRNA sequencing and untargeted metabolomics analyses. RESULTS: Our results showed that PSP exhibited significant anti-diabetic, anti-inflammatory and anti-oxidative effects on T2DM model rats. In addition, 16S rRNA sequencing showed that PSP treatment decreased the Firmicutes/ Bacteroidetes ratio in the gut. At the genus level, PSP treatment increased the relative abundances of Blautia, Adlercreutzia, Akkermansia and Parabacteroides while decreasing Prevotella, Megamonas funiformis and Escherichia. Untargeted metabolomics analysis revealed that PSP treatment could affect 20 metabolites, including hexanoylglycine, (±)5(6)-DiHET, ecgonine, L-cysteine-S-sulfate, epitestosterone, (±)12(13)-DiHOME, glutathione, L-ornithine, Dmannose 6-phosphate, L-fucose, L-tryptophan, L-kynurenine, serotonin, melatonin, 3-hydroxyanthranilic acid, xylitol, UDP-D-glucuronate, hydroxyproline, 4-guanidinobutyric acid, D-proline in T2DM model rats, these metabolites are associated with arginine and proline metabolism, tryptophan metabolism, amino sugar and nucleotide sugar metabolism, pentose and glucuronate interconversions, glutathione metabolism, arginine biosynthesis, ascorbate and aldarate metabolism pathways. Spearman correlation analysis results showed that the modulatory effects of PSP on the arginine and proline metabolism, tryptophan metabolism, and glutathione metabolism pathways were related to the regulation of Prevotella, Megamonas funiformis, Escherichia, Blautia and Adlercreutzia. CONCLUSION: Our research revealed the therapeutic, anti-inflammatory and anti-oxidative effects of PSP on T2DM. The mechanisms of PSP on T2DM are associated with improving the dysbiosis of gut microbiota and regulating arginine and proline metabolism, tryptophan metabolism, and glutathione metabolism in serum.

Synthesis and characterization of pseudocantharidins, novel phosphatase modulators that promote the inclusion of exon 7 into the SMN (survival of motoneuron) pre-mRNA.

Alternative pre-mRNA splicing is a central element of eukaryotic gene expression. Its deregulation can lead to disease, and methods to change splice site selection are developed as potential therapies. Spinal muscular atrophy is caused by the loss of the SMN1 (survival of motoneuron 1) gene. A therapeutic avenue for spinal muscular atrophy treatment is to promote exon 7 inclusion of the almost identical SMN2 (survival of motoneuron 2) gene. The splicing factor tra2-beta1 promotes inclusion of this exon and is antagonized by protein phosphatase (PP) 1. To identify new compounds that promote exon 7 inclusion, we synthesized analogs of cantharidin, an inhibitor of PP1, and PP2A. Three classes of compounds emerged from these studies. The first class blocks PP1 and PP2A activity, blocks constitutive splicing in vitro, and promotes exon 7 inclusion in vivo. The second class has no measurable effect on PP1 activity but activates PP2A. This class represents the first compounds described with these properties. These compounds cause a dephosphorylation of Thr-33 of tra2-beta1, which promotes exon 7 inclusion. The third class had no detectable effect on phosphatase activity and could promote exon 7 via allosteric effects. Our data show that subtle changes in similar compounds can turn a phosphatase inhibitor into an activator. These chemically related compounds influence alternative splicing by distinct mechanisms.

The snoRNA MBII-52 (SNORD 115) is processed into smaller RNAs and regulates alternative splicing.

The loss of HBII-52 and related C/D box small nucleolar RNA (snoRNA) expression units have been implicated as a cause for the Prader-Willi syndrome (PWS). We recently found that the C/D box snoRNA HBII-52 changes the alternative splicing of the serotonin receptor 2C pre-mRNA, which is different from the traditional C/D box snoRNA function in non-mRNA methylation. Using bioinformatic predictions and experimental verification, we identified five pre-mRNAs (DPM2, TAF1, RALGPS1, PBRM1 and CRHR1) containing alternative exons that are regulated by MBII-52, the mouse homolog of HBII-52. Analysis of a single member of the MBII-52 cluster of snoRNAs by RNase protection and northern blot analysis shows that the MBII-52 expressing unit generates shorter RNAs that originate from the full-length MBII-52 snoRNA through additional processing steps. These novel RNAs associate with hnRNPs and not with proteins associated with canonical C/D box snoRNAs. Our data indicate that not a traditional C/D box snoRNA MBII-52, but a processed version lacking the snoRNA stem is the predominant MBII-52 RNA missing in PWS. This processed snoRNA functions in alternative splice-site selection. Its substitution could be a therapeutic principle for PWS.

Er-Chen Decoction Alleviates High-Fat Diet-Induced Nonalcoholic Fatty Liver Disease in Rats through Remodeling Gut Microbiota and Regulating the Serum Metabolism.

Many studies have found that the dysfunction in gut microbiota and the metabolic dysfunction can promote nonalcoholic fatty liver disease (NAFLD) development. Er-Chen decoction (EC) can be used in the treatment of NAFLD. However, the mechanism of this hepatoprotection is still unknown. In this study, we constructed a rat model with NAFLD fed with high-fat chow and administered EC treatment. The therapeutic effects of EC on NAFLD were evaluated by measuring transaminases, blood lipid levels, and pathological changes in the liver. In addition, we measured the effects of EC on liver inflammatory response and oxidative stress. The changes in gut microbiota after EC treatment were studied using 16S rRNA sequencing. Serum untargeted metabolomics analysis was also used to study the metabolic regulatory mechanisms of EC on NAFLD. The results showed that EC decreased the serum transaminases and lipid levels and improved the pathological changes in NAFLD rats. Furthermore, EC enhanced the activities of SOD and GSH-Px and decreased MDA level in the liver. EC treatment also decreased the gene and protein levels of IL-6, IL-1ß, and TNF-α in the liver and serum. The 16S rRNA sequencing and untargeted metabolomics indicated that EC treatment affected the gut microbiota and regulated serum metabolism. Correlation analysis showed that the effects of EC on taurine and hypotaurine metabolism, cysteine and methionine metabolism, and vitamin B6 metabolism pathways were associated with affecting in the abundance of Lactobacillus, Dubosiella, Lachnospiraceae, Desulfovibri, Romboutsia, Akkermansia, Intestinimonas, and Candidatus_saccharimonas in the gut. In conclusion, our study confirmed the protective effect of EC on NAFLD. EC could treat NAFLD by inhibiting oxidative stress, reducing inflammatory responses, and improving the dysbiosis of gut microbiota and the modulation of the taurine and hypotaurine metabolism, cysteine and methionine metabolism, and vitamin B6 metabolism pathways in serum.

San-Huang-Yi-Shen Capsule Ameliorates Diabetic Kidney Disease through Inducing PINK1/Parkin-Mediated Mitophagy and Inhibiting the Activation of NLRP3 Signaling Pathway.

San-Huang-Yi-Shen capsule (SHYS) has been used in the treatment of diabetic kidney disease (DKD) in clinics. However, the mechanism of SHYS on DKD remains unclear. In this study, we used a high-fat diet combined with streptozocin (STZ) injection to establish a rat model of DKD, and different doses of SHYS were given by oral gavage to determine the therapeutic effects of SHYS on DKD. Then, we studied the effects of SHYS on PINK1/Parkin-mediated mitophagy and the activation of NLRP3 inflammasome to study the possible mechanisms of SHYS on DKD. Our result showed that SHYS could alleviate DKD through reducing the body weight loss, decreasing the levels of fasting blood glucose (FBG), and improving the renal function, insulin resistance (IR), and inhibiting inflammatory response and oxidative stress in the kidney. Moreover, transmission electron microscopy showed SHYS treatment improved the morphology of mitochondria in the kidney. In addition, western blot and immunoflourescence staining showed that SHYS treatment induced the PINK1/Parkin-mediated mitophagy and inhibited the activation of NLRP3 signaling pathway. In conclusion, our study demonstrated the therapeutic effects of SHYS on DKD. Additionally, our results indicated that SHYS promoted PINK1/Parkin-mediated mitophagy and inhibited NLRP3 inflammasome activation to improve mitochondrial injury and inflammatory responses.

Baihu renshen decoction ameliorates type 2 diabetes mellitus in rats through affecting gut microbiota enhancing gut permeability and inhibiting TLR4/NF-κB-mediated inflammatory response.

Baihu Rensheng decoction (BHRS) can effectively improve insulin resistance (IR) and decrease blood glucose in diabetic patients. However, its specific mechanism of action remains unclear. In this study, a type 2 diabetes mellitus (T2DM) rat model was established using a high-fat diet combined with streptozotocin (STZ) injection and treated with BHRS. Firstly, the therapeutic and anti-inflammatory effects of BHRS on T2DM were evaluated. Secondly, the effects of BHRS on gut permeability were evaluated and western blot was used to detect the changes of TLR4/NF-κB pathway-related protein expressions in liver. Finally, 16S rRNA sequencing was used to detect alteration of gut microbiota diversity and abundance in rats after BHRS treatment. Our results showed that BHRS could alleviate the hyperglycemia, hyperlipidemia, IR, and pathological changes of liver, pancreas, and kidney in T2DM rats. BHRS could also decrease the levels of pro-inflammatory cytokines and inhibit the oxidative stress. Immunohistochemistry showed BHRS could increase the expression tight junction-related proteins (ZO-1 and occludin) in colon. Besides, the level of LPS in serum was decreased after BHRS treatment. Western blot results showed that the protein expression of TLR4, MyD88 and the phosphorylation IκB, and NF-κBp65 were lowered after BHRS treatment. 16S rRNA sequencing showed that BHRS treatment altered the diversity of gut microbiotra and decreases the Firmicutes/Bacteroidetes (F to B) ratio at the phylum level. At the genus level, BHRS could increase the relative abundances of Lactobacillus, Blautia, and Anaerostipes and decrease the relative abundances of Allobaculum, Candidatus Saccharimonas, and Ruminococcus. In conclusion, our study revealed the various ameliorative effects of BHRS on T2DM, including improving the liver and kidney functions and alleviating the hyperglycemia, hyperlipidemia, pathological changes, oxidative stress and inflammatory response. The mechanisms of BHRS on T2DM are likely linked to the repair of gut barrier and the inhibition of TLR4/NF-κB-mediated inflammatory response and the improvement in the dysbiosis of gut microbiota.

The YTH domain is a novel RNA binding domain.

The YTH (YT521-B homology) domain was identified by sequence comparison and is found in 174 different proteins expressed in eukaryotes. It is characterized by 14 invariant residues within an alpha-helix/beta-sheet structure. Here we show that the YTH domain is a novel RNA binding domain that binds to a short, degenerated, single-stranded RNA sequence motif. The presence of the binding motif in alternative exons is necessary for YT521-B to directly influence splice site selection in vivo. Array analyses demonstrate that YT521-B predominantly regulates vertebrate-specific exons. An NMR titration experiment identified the binding surface for single-stranded RNA on the YTH domain. Structural analyses indicate that the YTH domain is related to the pseudouridine synthase and archaeosine transglycosylase (PUA) domain. Our data show that the YTH domain conveys RNA binding ability to a new class of proteins that are found in all eukaryotic organisms.

Pi-Dan-Jian-Qing Decoction Ameliorates Type 2 Diabetes Mellitus Through Regulating the Gut Microbiota and Serum Metabolism.

Pi-Dan-Jian-Qing decoction (PDJQ) can been used in the treatment of type 2 diabetes mellitus (T2DM) in clinic. However, the protective mechanisms of PDJQ on T2DM remain unknown. Recent studies have shown that the changes in gut microbiota could affect the host metabolism and contribute to progression of T2DM. In this study, we first investigated the therapeutic effects of PDJQ on T2DM rats. 16S rRNA sequencing and untargeted metabolomics analyses were used to investigate the mechanisms of action of PDJQ in the treatment of T2DM. Our results showed that PDJQ treatment could improve the hyperglycemia, hyperlipidemia, insulin resistance (IR) and pathological changes of liver, pancreas, kidney, and colon in T2DM rats. PDJQ could also decrease the levels of pro-inflammatory cytokines and inhibit the oxidative stress. 16S rRNA sequencing showed that PDJQ could decrease the Firmicutes/Bacteroidetes (F to B) ratio at the phylum level. At the genus level, PDJQ could increase the relative abundances of Lactobacillus, Blautia, Bacteroides, Desulfovibrio and Akkermansia and decrease the relative abundance of Prevotella. Serum untargeted metabolomics analysis showed that PDJQ could regulate tryptophan metabolism, histidine metabolism, tricarboxylic acid (TCA) cycle, phenylalanine, tyrosine and tryptophan biosynthesis and tyrosine metabolism pathways. Correlation analysis indicated that the modulatory effects of PDJQ on the tryptophan metabolism, histidine metabolism and TCA cycle pathways were related to alterations in the abundance of Lactobacillus, Bacteroides and Akkermansia. In conclusion, our study revealed the various ameliorative effects of PDJQ on T2DM, including improving the liver and kidney functions and alleviating the hyperglycemia, hyperlipidemia, IR, pathological changes, oxidative stress and inflammatory response. The mechanisms of PDJQ on T2DM are likely linked to an improvement in the dysbiosis of gut microbiota and modulation of tryptophan metabolism, histamine metabolism, and the TCA cycle.

Jian-Gan-Xiao-Zhi Decoction Alleviates Inflammatory Response in Nonalcoholic Fatty Liver Disease Model Rats through Modulating Gut Microbiota.

BACKGROUND: Jian-Gan-Xiao-Zhi decoction (JGXZ), composed of Salvia miltiorrhiza Bunge, Panax notoginseng, Curcuma zedoaria, and other 9 types of herbs, has demonstrated beneficial effects on nonalcoholic fatty liver disease (NAFLD). However, the mechanisms behind JGXZ's impact on NAFLD remain unknown. METHODS: In this study, a NAFLD rat model induced by a high-fat diet (HFD) received oral treatment of JGXZ (8 or 16 g crude herb/kg) for 12 weeks. The therapeutic effects of JGXZ on NAFLD model rats were investigated through blood lipid levels and pathological liver changes. 16S rRNA analysis was used to study the changes in gut microbiota after JGXZ treatment. The expressions of occludin and tight junction protein 1 (ZO-1) in the colon were investigated using immunostaining to study the effects of JGXZ on gut permeability. The anti-inflammatory effects of JGXZ were also studied through measuring the levels of IL-1ß, IL-6, and TNF-α in the serum and liver. RESULTS: JGXZ treatment could decrease body weight and ameliorate dyslipidemia in NAFLD model rats. H&E and Oil Red O staining indicated that JGXZ reduced steatosis and infiltration of inflammatory cells in the liver. 16S rRNA analysis showed that JGXZ impacted the diversity of gut microbiota, decreasing the Firmicutes-to-Bacteroidetes ratio, and increasing the relative abundance of probiotics, such as Alloprevotella, Lactobacillus, and Turicibacter. Gut permeability evaluation found that the expressions of ZO-1 and occludin in the colon were increased after JGXZ treatment. Moreover, JGXZ treatment could decrease the levels of IL-1ß, IL-6, and TNF-α in the serum and liver. CONCLUSIONS: Our study illustrated that JGXZ could ameliorate NAFLD through modulating gut microbiota, decreasing gut permeability, and alleviating inflammatory response.

Ethanol extract of Ardisiae Japonicae Herba inhibits hepatoma carcinoma cell proliferation in vitro through regulating lipid metabolism.

Objective: The aim of this study is to discover the possible working mechanisms of Ardisiae Japonicae Herba (AJH) on hepatoma carcinoma (HCC). Methods: In this study, ethanol extract of AJH was prepared and used to treat HCC cell in vitro. Furthermore, a genomic wide RNA sequencing (RNA-seq) was performed to screen deregulated genes in HCC cells after the treatment of AJH extract. The gene and protein expression related to lipid metabolism in HCC cells were also investigated to validate the results obtained from RNA-seq. Results: AJH extract could inhibit HCC cell proliferation in vitro. RNA-seq analysis has identified 1,601 differentially expressed genes (DEGs, fold change ≥ 2.0 or fold change ≤ 0.5, P < 0.05) in HCC after AJH extract treatment, which included 225 up-regulated genes and 1,376 down-regulated genes. KEGG pathway analysis of DEGs demonstrated that lipid metabolism was a potential pathway related to AJH treatment. In agreement with the RNA-seq data, qPCR and Western-blot analysis indicated that expression of genes and proteins related to lipid metabolism (SREBP1, ACC, ACLY and FASN) were significantly down-regulated in AJH treatment group as compared with the control group. Furthermore, AJH extract could also decrease lipid contents and cellular free fatty acid levels in HCC cells. Conclusion: Ethanol extract of AJH could inhibit HCC cell proliferation in vitro, the possible mechanism may be related to the inhibition of lipid metabolism.

Pre-mRNA secondary structures influence exon recognition.

The secondary structure of a pre-mRNA influences a number of processing steps including alternative splicing. Since most splicing regulatory proteins bind to single-stranded RNA, the sequestration of RNA into double strands could prevent their binding. Here, we analyzed the secondary structure context of experimentally determined splicing enhancer and silencer motifs in their natural pre-mRNA context. We found that these splicing motifs are significantly more single-stranded than controls. These findings were validated by transfection experiments, where the effect of enhancer or silencer motifs on exon skipping was much more pronounced in single-stranded conformation. We also found that the structural context of predicted splicing motifs is under selection, suggesting a general importance of secondary structures on splicing and adding another level of evolutionary constraints on pre-mRNAs. Our results explain the action of mutations that affect splicing and indicate that the structural context of splicing motifs is part of the mRNA splicing code.

The Anti-Depressive Effects of Hesperidin and the Relative Mechanisms Based on the NLRP3 Inflammatory Signaling Pathway.

There is increasing evidence showing that inflammation is associated with depression in humans. Hesperidin, a natural bioflavonoid, has performed excellent effects on depression. The aim of this research was to investigate the therapeutic effect of hesperidin on chronic unpredictable mild stress (CUMS)-induced rats. The sucrose preference test (SPT), forced swimming test (FST), and open field test (OFT) were performed to measure the depression-related symptoms. The enzyme-linked immunosorbent assay (ELISA) was used to determine the concentrations of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in the prefrontal cortex (PFC) of rats and cellular supernatant. PCR and Western blot were used to monitor the differences of NLRP3, caspase-1, ASC activation in the levels of genes and proteins in the PFC of rats and microglia. The activation of microglia was determined using immunofluorescence staining and flow cytometry assay. Our results show that hesperidin treatment significantly relieved depressive like behaviors in CUMS rats. In addition, hesperidin decreased the expression levels of IL-1ß, IL-6, TNF-α, NLRP3, caspase-1, and ASC in the PFC and microglia. This study investigated that hesperidin treatment ameliorated CUMS-induced depression by suppressing microglia and inflammation.

Kang-Xian Pills Inhibit Inflammatory Response and Decrease Gut Permeability to Treat Carbon Tetrachloride-Induced Chronic Hepatic Injury through Modulating Gut Microbiota.

Kang-Xian (KX) pills have been clinically used for the treatment of chronic hepatic injury (CHI). However, the mechanisms of KX on CHI remain unknown. The aim of this study mainly focused on the anti-inflammatory effects of KX in a CHI mouse model based on modulating gut microbiota and gut permeability. We first established a CHI model using carbon tetrachloride (CCl4) and treated it with KX. The anti-inflammatory effects of KX on CHI model mice and the changes in gut permeability after KX treatment were also investigated. 16S rRNA analysis was used to study the changes of gut microbiota composition after KX treatment. In addition, gut microbiota was depleted using a combination of antibiotics in order to further confirm that KX could inhibit the inflammatory response and decrease gut permeability to treat CHI by modulating the gut microbiota. Results showed that KX treatment significantly improved liver function in CHI model mice. KX could also increase the levels of tight junction proteins in the colon and decrease the expression of proinflammatory cytokines in the liver. 16S rRNA analysis indicated that KX treatment affected the alpha and beta diversities in CHI model mice. Further analysis of 16S rRNA sequencing indicated that KX treatment increased the ratio of Firmicutes to Bacteroidetes at the phylum level. At the genus level, KX treatment increased the relative abundance of Lactobacillus, Bacteroides, and Akkermansia and decreased the relative abundance of Ralstonia, Alloprevotella, and Lachnoclostridium. However, KX could not alleviate CHI after depleting the gut microbiota. The effects of KX on gut permeability and inflammatory response in the liver were also decreased following the depletion of gut microbiota. In conclusion, our current study demonstrated that gut microbiota was significantly affected during CHI progression. KX could inhibit the inflammatory response and decrease the gut permeability in CHI model mice through modulating the gut microbiota.

Da-Chai-Hu Decoction Ameliorates High Fat Diet-Induced Nonalcoholic Fatty Liver Disease Through Remodeling the Gut Microbiota and Modulating the Serum Metabolism.

The dysbiosis in gut microbiota could affect host metabolism and contribute to the development of nonalcoholic fatty liver disease (NAFLD). Da-Chai-Hu decoction (DCH) has demonstrated protective effects on NAFLD, however, the exact mechanisms remain unclear. In this study, we established a NAFLD rat model using a high fat diet (HFD) and provided treatment with DCH. The changes in gut microbiota post DCH treatment were then investigated using 16S rRNA sequencing. Additionally, serum untargeted metabolomics were performed to examine the metabolic regulations of DCH on NAFLD. Our results showed that DCH treatment improved the dyslipidemia, insulin resistance (IR) and ameliorated pathological changes in NAFLD model rats. 16S rRNA sequencing and untargeted metabolomics showed significant dysfunction in gut microbiota community and serum metabolites in NAFLD model rats. DCH treatment restored the dysbiosis of gut microbiota and improved the dysfunction in serum metabolism. Correlation analysis indicated that the modulatory effects of DCH on the arachidonic acid (AA), glycine/serine/threonine, and glycerophospholipid metabolic pathways were related to alterations in the abundance of Romboutsia, Bacteroides, Lactobacillus, Akkermansia, Lachnoclostridium and Enterobacteriaceae in the gut microflora. In conclusion, our study revealed the ameliorative effects of DCH on NAFLD and indicated that DCH's function on NAFLD may link to the improvement of the dysbiosis of gut microbiota and the modulation of the AA, glycerophospholipid, and glycine/serine/threonine metabolic pathways.