The Study of Yin-Chen-Hao-Tang Preventing and Treating Alcoholic Fatty Liver Disease through PPAR Signaling Pathway Based on Network Pharmacology and RNA-Seq Transcriptomics

Background. Alcoholic fatty liver disease (AFLD) is the first stage of the alcoholic liver disease course. Yin-Chen-Hao-Tang (YCHT) has a good clinical effect on the treatment of AFLD, but its molecular mechanism has not been elucidated. In this study, we tried to explore the molecular mechanism of YCHT in improving hepatocyte steatosis in AFLD mice through network pharmacology and RNA sequencing (RNA-Seq) transcriptomics. Methods. Network pharmacological methods were used to analyze the potential therapeutic signaling pathways and targets of YCHT on AFLD. Then, the AFLD mice model was induced and YCHT was administered concurrently. Liver injury was measured by serum alanine aminotransferase (ALT) activity and liver tissue H&E staining, and liver steatosis was determined by serum triglyceride (TG) level and liver tissue Oil Red staining. The molecular mechanism of YCHT on prevention and treatment of mice AFLD was investigated according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differential expression genes data obtained by liver tissue RNA-Seq. Finally, ethanol-induced AFLD AML12 hepatocyte model was established, YCHT with or without PPARα agonist pemafibrate or PPARγ inhibitor GW9662 was administered, Nile Red fluorescent staining was used to evaluate steatosis levels in AML12 hepatocytes, and qRT-PCR was used to detect PPARα and PPARγ gene expression. Results. The results of network pharmacology analysis showed that YCHT may exert its pharmacological effect on AFLD through 312 potential targets which are involved in many signaling pathways including the PPAR signaling pathway. AFLD mice experiments results showed that YCHT markedly decreased mice serum ALT activity and serum TG levels. YCHT also significantly improved alcohol-induced hepatic injury and steatosis in mice livers. Furthermore, KEGG pathway enrichment results of RNA-Seq showed that the PPAR signaling pathway should be the most relevant pathway of YCHT in the prevention and treatment of AFLD. AFLD hepatocyte model experiment results showed that YCHT could remarkably reduce hepatocyte steatosis through reducing PPARγ expression and increasing PPARα expression. Conclusions. Our study discovered that PPARγ and PPARα are the key targets and the PPAR signaling pathway is the main signaling pathway for YCHT to prevent and treat AFLD.

Lipopolysaccharide-Induced Transcriptional Changes in LBP-Deficient Rat and Its Possible Implications for Liver Dysregulation during Sepsis

Sepsis is an organ dysfunction caused by the dysregulated inflammatory response to infection. Lipopolysaccharide-binding protein (LBP) binds to lipopolysaccharide (LPS) and modulates the inflammatory response. A rare systematic study has been reported to detect the effect of LBP gene during LPS-induced sepsis. Herein, we explored the RNA sequencing technology to profile the transcriptomic changes in liver tissue between LBP-deficient rats and WT rats at multiple time points after LPS administration. We proceeded RNA sequencing of liver tissue to search differentially expressed genes (DEGs) and enriched biological processes and pathways between WT and LBP-deficient groups at 0 h, 6 h, and 24 h. In total, 168, 284, and 307 DEGs were identified at 0 h, 6 h, and 24 h, respectively, including Lrp5, Cyp7a1, Nfkbiz, Sigmar1, Fabp7, and Hao1, which are related to the inflammatory or lipid-related process. Functional enrichment analysis revealed that inflammatory response to LPS mediated by Ifng, Cxcl10, Serpine1, and Lbp was enhanced at 6 h, while lipid-related metabolism associated with C5, Cyp4a1, and Eci1 was enriched at 24 h after LPS administration in the WT samples. The inflammatory process was not found when the LBP gene was knocked out; lipid-related metabolic process and peroxisome proliferator-activated receptor (PPAR) signaling pathway mediated by Dhrs7b and Tysnd1 were significantly activated in LBP-deficient samples. Our study suggested that the invading LPS may interplay with LBP to activate the nuclear factor kappa B (NF-κB) signaling pathway and trigger uncontrolled inflammatory response. However, when inhibiting the activity of NF-κB, lipid-related metabolism would make bacteria removal via the effect on the PPAR signaling pathway in the absence of LBP gene. We also compared the serum lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) levels using the biochemistry analyzer and analyzed the expression of high mobility group box 1 (HMGB1) and cleaved-caspase 3 with immunohistochemistry, which further validated our conclusion.

Tissue-Specific Expression Pattern in Ancherythroculter nigrocauda, a Sexually Size Dimorphic Fish

Certain members of the Actinopterygii class are known to exhibit sexual dimorphism (SD) that results in major phenotypic differences between male and female fishes of a species. One of the most common differences between the two sexes is in body weight, a factor with a high economic value in aquaculture. In this study, we used RNA sequencing (RNA-seq) to study the liver and brain transcriptomes of Ancherythroculter nigrocauda, a fish exhibiting SD. Females attain about fourfold body weight of males at sexual maturity. Sample clustering showed that both sexes were grouped well with their sex phenotypes. In addition, 2,395 and 457 differentially expressed genes (DEGs) were identified in the liver and brain tissues, respectively. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses predicted the association of PPAR signaling, cytochrome P450, and steroid hormone biosynthesis to the differences in sexual size. In addition, weighted gene co-expression network analyses (WGCNA) were conducted, and the green module was identified to be significantly correlated with sexual size dimorphism (SSD). Altogether, these results improve our understanding of the molecular mechanism underlying SSD in A. nigrocauda.

Identification of biological processes and signaling pathways for the knockout of REV-ERB in mouse brain

REV-ERB is an orphan nuclear receptor that is widely expressed in the brain and inhibits transcriptional activities. A variety of genes affect the activity and expression of REV-ERB. In this study, our objective is to identify significant signaling pathways and biological processes in the knockout of the REV-ERB mouse brain. The GSE152919 dataset was originally created by using the Illumina HiSeq 4000 (Mus musculus). The KEGG and GO analyses suggested that biological processes "PPAR signaling", "Hippo signaling", and "Hypertrophic cardiomyopathy (HCM)" are mostly affected in the knockout of REV-ERB. Furthermore, we identified a number of genes according to the PPI network including NPAS2, CRY2, BMAL1, and CRY1 which were involved in the lack of REV-ERB in the brain. Therefore, our study provides further insights into the study of circadian clocks.

Comprehensive Analysis of the Transcriptome-Wide m6A Methylation Modification Difference in Liver Fibrosis Mice by High-Throughput m6A Sequencing

N6-Methyladenosine (m6A), a unique and common mRNA modification method in eukaryotes, is involved in the occurrence and development of many diseases. Liver fibrosis (LF) is a common response to chronic liver injury and may lead to cirrhosis and even liver cancer. However, the involvement of m6A methylation in the development of LF is still unknown. In this study, we performed a systematic evaluation of hepatic genome-wide m6A modification and mRNA expression by m6A-seq and RNA-seq using LF mice. There were 3,315 genes with significant differential m6A levels, of which 2,498 were hypermethylated and 817 hypomethylated. GO and KEGG analyses illustrated that differentially expressed m6A genes were closely correlated with processes such as the endoplasmic reticulum stress response, PPAR signaling pathway and TGF-β signaling pathway. Moreover, a total of 90 genes had both a significant change in the m6A level and mRNA expression shown by joint analysis of m6A-seq and RNA-seq. Hence, the critical elements of m6A modification, including methyltransferase WTAP, demethylases ALKBH5 and binding proteins YTHDF1 were confirmed by RT-qPCR and Western blot. In an additional cell experiment, we also observed that the decreased expression of WTAP induced the development of LF as a result of promoting hepatic stellate cell (HSC) activation. Therefore, this study revealed unique differential m6A methylation patterns in LF mice and suggested that m6A methylation was associated with the occurrence and course of LF to some extent.

Trait correlated expression combined with eQTL and ASE analyses identified novel candidate genes affecting intramuscular fat

Background Intramuscular fat (IMF) content is a determining factor for meat taste. The Luchuan pig is a fat-type local breed in southern China that is famous for its desirable meat quality due to high IMF, however, the crossbred offspring of Luchuan sows and Duroc boars displayed within-population variation on meat quality, and the reason remains unknown. Results In the present study, we identified 212 IMF-correlated genes (FDR ≤ 0.01) using correlation analysis between gene expression level and the value of IMF content. The IMF-correlated genes were significantly enriched in the processes of lipid metabolism and mitochondrial energy metabolism, as well as the AMPK/PPAR signaling pathway. From the IMF-correlated genes, we identified 99 genes associated with expression quantitative trait locus (eQTL) or allele-specific expression (ASE) signals, including 21 genes identified by both cis-eQTL and ASE analyses and 12 genes identified by trans-eQTL analysis. Genome-wide association study (GWAS) of IMF identified a significant QTL on SSC14 (p-value = 2.51E−7), and the nearest IMF-correlated gene SFXN4 (r = 0.28, FDR = 4.00E−4) was proposed as the candidate gene. Furthermore, we highlighted another three novel IMF candidate genes, namely AGT, EMG1, and PCTP, by integrated analysis of GWAS, eQTL, and IMF-gene correlation analysis. Conclusions The AMPK/PPAR signaling pathway together with the processes of lipid and mitochondrial energy metabolism plays a vital role in regulating porcine IMF content. Trait correlated expression combined with eQTL and ASE analysis highlighted a priority list of genes, which compensated for the shortcoming of GWAS, thereby accelerating the mining of causal genes of IMF.

Developmental Programming: Prenatal Testosterone Excess on Liver and Muscle Coding and Non-Coding RNA in Female Sheep

Prenatal testosterone (T)-treated female sheep manifest peripheral insulin resistance, ectopic lipid accumulation and insulin signaling disruption in liver and muscle. This study investigated transcriptional changes and transcriptome signature of prenatal T excess-induced hepatic and muscle-specific metabolic disruptions. Genome-wide coding and non-coding (nc) RNA expression in liver and muscle from 21-month-old prenatal T-treated (T propionate 100mg intramuscular twice weekly from days 30 to 90 of gestation; Term: 147 days) and control females were compared. Prenatal T (1) induced differential expression of mRNAs in liver (15 down, 17 up) and muscle (66 down, 176 up) (FDR<0.05, absolute log2 fold change>0.5); (2) downregulated mitochondrial pathway genes in liver and muscle; (3) downregulated hepatic lipid catabolism and PPAR signaling gene pathways; (4) modulated ncRNA metabolic processes gene pathway in muscle and (5) downregulated 5 uncharacterized long ncRNA (lncRNA) in the muscle but no ncRNA changes in the liver. Correlation analysis showed downregulation of lncRNAs LOC114112974 and LOC105607806 was associated with decreased TPK1, and LOC114113790 with increased ZNF470 expression. Orthogonal Projections to Latent Structures Discriminant Analysis identified mRNAs HADHA and SLC25A45, and miRNAs MIR154A, MIR25 and MIR487B in liver and ARIH1 and ITCH and miRNAs MIR369, MIR10A and MIR10B in muscle as potential biomarkers of prenatal T-excess. These findings suggest downregulation of mitochondria, lipid catabolism, and PPAR signaling genes in liver and dysregulation of mitochondrial and ncRNA gene pathways in muscle are contributors of lipotoxic and insulin resistant hepatic and muscle phenotype. Gestational T excess programming of metabolic dysfunctions involve tissue-specific ncRNA modulated transcriptional changes.

Dynamic Microbial Shifts and Signatures of Long-Term Remission in Allergic Rhinitis After an Herbal Formula Treatment

A mixed Chinese herbal formula, Xiao-Qing-Long-Decoction (XQLD), may contribute to sustained remission in allergic rhinitis (AR), but it is unknown which factors determine such long-term effect. Here, we aimed to identify bacterial signatures associated with sustained remission. To this end, samples from AR patients at four different times were analyzed to compare the dynamic bacterial community and structure shifts. Diversity indices Chao1 showed significant difference across different time (p<0.05), and the Kruskal-Wallis test identified that Dialister (OTU_31), Roseburia (OTU_36), Bacteroides (OTU_22), Bacteroides (OTU_2040), and Prevotella_9 (OTU_5) were the significant differential bacterial taxa (p<0.05). These distinctive genera were significantly associated with the change of AR clinical indices and the predicted functional pathways such as PPAR signaling pathway, peroxisome, and citrate cycle (TCA cycle) (p<0.05), indicating that they may be important bacterial signatures involving in the sustained remission in AR (p<0.05). Besides, lower Firmicutes/Bacteroidetes (F/B) ratio at 6 months follow-up may also contribute to the long-term remission of AR. No seriously adverse events and safety concerns were observed in this study. In conclusion, XQLD is a meaningful, long-term efficient and safe medication for AR treatment. The underlying mechanisms of sustained remission in AR after XQLD treatment may be associated with the dynamic alteration of featured gut bacteria taxa.

Atorvastatin Ester Regulates Lipid Metabolism in Hyperlipidemia Rats via the PPAR-Signaling Pathway and HMGCR Expression in the Liver

Atorvastatin ester (Ate) is a structural trim of atorvastatin that can regulate hyperlipidemia. The purpose of this study was to evaluate the lipid-lowering effect of Ate. Male Sprague Dawley (SD) rats were fed a high-fat diet for seven months and used as a hyperlipidemia model. The lipid level and liver function of the hyperlipidemia rats were studied by the levels of TG, TC, LDL, HDL, ALT, and AST in serum after intragastric administration with different doses of Ate. HE staining was used to observe the pathological changes of the rat liver and gastrocnemius muscle. The lipid deposits in the liver of rats were observed by staining with ORO. The genes in the rat liver were sequenced by RNA-sequencing. The results of the RNA-sequencing were further examined by qRT-PCR and western blotting. Biochemical test results indicated that Ate could obviously improve the metabolic disorder and reduce both the ALT and AST levels in serum of the hyperlipidemia rats. Pathological results showed that Ate could improve HFD-induced lipid deposition and had no muscle toxicity. The RNA-sequencing results suggested that Ate affected liver lipid metabolism and cholesterol, metabolism in the hyperlipidemia-model rats may vary via the PPAR-signaling pathway. The western blotting and qRT-PCR results demonstrated the Ate-regulated lipid metabolism in the hyperlipidemia model through the PPAR-signaling pathway and HMGCR expression. In brief, Ate can significantly regulate the blood lipid level of the model rats, which may be achieved by regulating the PPAR-signaling pathway and HMGCR gene expression.