Combining network pharmacology with chromatographic fingerprinting and multicomponent quantitative analysis for the quality evaluation of Moutan Cortex.

In this study, we combined network pharmacology, chromatographic fingerprinting and multicomponent quantitative analysis to evaluate the quality of Moutan Cortex (MC). Specifically, through network pharmacology, we obtained a comprehensive understanding of the active components and pharmacological activities of MC. In addition, the method of establishing fingerprint and multicomponent quantification by ultra high-performance liquid chromatography is convenient and comprehensive, and can more fully reflect the overall distribution of various chemical components. Principal component analysis and discriminant least square analysis were used. The results show that MC plays a synergistic role through multiple targets and pathways. The contents of chemical components in MC from different sources were significantly different. Combining network pharmacology and multicomponent quantitative results, gallic acid, benzoyl paeonol, paeonol, methyl gallate, benzoic acid and paeonol can be used as quality markers for MC quality control. This study provides a comprehensive and reliable strategy for the quality evaluation of MC and determines its quality indicators to ensure the quality of Chinese herbal medicine.

Dihydroartemisinin improves hypercholesterolemia in ovariectomized mice via enhancing vectorial transport of cholesterol and bile acids from blood to bile.

The increase of concentrations of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in the serum of postmenopausal women is the important risk factor of the high morbidity of cardiovascular diseases of old women worldwide. To test the anti-hypercholesterolemia function of dihydroartemisinin (DHA) in postmenopausal women, ovariectomized (OVX) mice were generated, and DHA were administrated to OVX mice for 4 weeks. The blood and liver tissues were collected for biochemical and histological tests respectively. The mRNA and protein expression levels of genes related to metabolism and transport of cholesterol, bile acid and fatty acid in the liver or ileum were checked through qPCR and western blot. DHA could significantly reduce the high concentrations of TC and LDL-C in the serum and the lipid accumulation in the liver of ovariectomized mice. The expression of ABCG5/8 was reduced in liver of OVX mice, and DHA could up-regulate the expression of them. Genes of transport proteins for bile salt transport from blood to bile, including Slc10a1, Slco1b2 and Abcb11, were also significantly up-regulated by DHA. DHA also down-regulated the expression of Slc10a2 in the ileum of OVX mice to reduce the absorption of bile salts. Genes required for fatty acid synthesis and uptake, such as Fasn and CD36, were reduced in the liver of OVX mice, and DHA administration could significantly up-regulate the expression of them. These results demonstrated that DHA could improve hypercholesterolemia in OVX mice through enhancing the vectorial transport of cholesterol and bile acid from blood to bile.

Ovariectomy Impaired Hepatic Glucose and Lipid Homeostasis and Altered the Gut Microbiota in Mice With Different Diets.

The lower incidence of metabolic diseases of women than men and the increasing morbidity of metabolic disorders of menopausal women indicated that hormones produced by ovaries may affect homeostasis of glucose and lipid metabolism, but the underlying mechanisms remain unclear. To explore the functions of ovaries on regulating glucose and lipid metabolism in females, 8 weeks old C57BL/6 mice were preformed ovariectomy and administrated with normal food diet (NFD) or high fat diet (HFD). Six weeks after ovariectomy, blood biochemical indexes were tested and the morphology and histology of livers were checked. The expression levels of genes related to glucose and lipid metabolism in liver were detected through transcriptome analysis, qPCR and western blot assays. 16S rDNA sequence was conducted to analyze the gut microbiota of mice with ovariectomy and different diets. The serum total cholesterol (TC) was significantly increased in ovariectomized (OVX) mice fed with NFD (OVXN), and serum low density lipoprotein-cholesterol (LDL-C) was significantly increased in both OVXN mice and OVX mice fed with HFD (OVXH). The excessive glycogen storage was found in livers of 37.5% mice from OVXN group, and lipid accumulation was detected in livers of the other 62.5% OVXN mice. The OVXN group was further divided into OVXN-Gly and OVXN-TG subgroups depending on histological results of the liver. Lipid drops in livers of OVXH mice were more and larger than other groups. The expression level of genes related with lipogenesis was significantly increased and the expression level of genes related with ß-oxidation was significantly downregulated in the liver of OVXN mice. Ovariectomy also caused the dysbiosis of intestinal flora of OVXN and OVXH mice. These results demonstrated that hormones generated by ovaries played important roles in regulating hepatic glucose and lipid metabolism and communicating with the gut microbiota in females.

High dose lithium chloride causes colitis through activating F4/80 positive macrophages and inhibiting expression of Pigr and Claudin-15 in the colon of mice.

OBJECTIVE: Lithium chloride (LiCl) was a mood stabilizer for bipolar affective disorders and it could activate Wnt/ß-catenin signaling pathway both in vivo and in vitro. Colon is one of a very susceptible tissues to Wnt signaling pathway, and so it would be very essential to explore the toxic effect of a high dose of LiCl on colon. METHODS: C57BL/6 mice were injected intraperitoneally with 200 mg/kg LiCl one dose a day for 5 days to activate Wnt signal pathway in intestines. H&E staining was used to assess the colonic tissues of mice treated with high dose of LiCl. The expression of inflammation-associated genes and tight junction-associated genes in colons was measured using qPCR, Western blot and immunostaining methods. The gut microbiome was tested through 16S rDNA gene analysis. RESULTS: The differentiation of enteroendocrine cells in colon was inhibited by treatment of 200 mg/kg LiCl. The F4/80 positive macrophages in colon were activated by high dose of LiCl, and migrated from the submucosa to the lamina propria. The expression of pro-inflammatory genes TNFα and IL-1ß was increased in the colon of high dose of LiCl treated mice. Clostridium_sp_k4410MGS_306 and Prevotellaceae_UCG_001 were specific and predominant for the high dose of LiCl treated mice. The expression of IgA coding genes, Pigr and Claudin-15 was significantly decreased in the colon tissues of the high dose of LiCl treated mice. CONCLUSION: 200 mg/kg LiCl might cause the inflammation in colon of mice through activating F4/80 positive macrophages and inhibiting the expression of IgA coding genes in plasma cells and the expression of Pigr and Claudin-15 in colonic epithelial cells, providing evidences for the toxic effects of high dose of LiCl on colon.

EpCAM is essential for maintenance of the small intestinal epithelium architecture via regulation of the expression and localization of proteins that compose adherens junctions.

Epithelial cell adhesion molecule (EpCAM) is highly expressed in mammalian intestines, and is essential for maintaining the homeostasis of the intestinal epithelium. EpCAM protein is localized at tight junctions and the basolateral membrane of the intestinal epithelium, where it interacts with many cell adhesion molecules. To explore the molecular functions of EpCAM in regulating adherens junctions in the intestinal epithelium, EpCAM knockout embryos and newborn pups were analyzed. Hematoxylin and eosin staining was used to assess the histology of the duodenum, jejunum, ileum and colon from wild-type and EpCAM­/­ mice at E18.5, P0 and P3. The expression and localization of adherens junction­associated genes and genes that encode the proteins that participate in the assembly of adherens junctions were measured at the mRNA and protein levels using qPCR, western blot analysis and immunofluorescence staining. The results showed that although there was no significant damage to the intestines of EpCAM­/­ mice at E18.5 and P0, they were significantly damaged at P3 in mutant mice. The expression of adherens junction­associated genes in EpCAM mutant mice was normal at the mRNA level from E18.5 to P3, but their protein levels were gradually reduced and mislocalized from E18.5 to P3. The expression of nectin 1, which can regulate the assembly and adhesion activity of E­cadherin, was also gradually reduced at both the mRNA and protein levels in the intestinal epithelium of EpCAM mutant mice from E18.5 to P3. In summary, the loss of EpCAM may cause the reduction and mislocalization of proteins that compose adherens junctions partly via the downregulation of nectin 1 in the intestines.

Cigarette smoking exposure breaks the homeostasis of cholesterol and bile acid metabolism and induces gut microbiota dysbiosis in mice with different diets.

Exposure of humans to second-hand smoking (SHS) increases glucose and lipid metabolic disorders. The link of hepatic metabolic dysfunction to environmental cigarette smoking has been noticed, but the related mechanism is still unclear. C57BL/6 mice with normal food diet (NFD) or high fat diet (HFD) were exposed to 15 min cigarette smoking twice a day in a 0.038 m3 box for 4 weeks, and the concentration of nicotine in the air of the box was 21.05 mg/m3 during the smoke exposure. Liver tissues and serum were collected for gene expression and biochemistry test. The fecal microbiota was also checked through 16S rDNA sequences. Cigarette smoking exposure increased the accumulation of total cholesterol (TC) in liver, and the expression of cholesterol synthesis-related genes was upregulated. The expression of CYP8B1 protein was significantly down-regulated, and the ratio of cholic acid (CA) to chenodeoxycholic acid (CDCA) was significantly reduced in the liver of mice exposed to cigarette smoking especially for HFD group. Cigarette smoking exposure caused insulin resistance in the liver of mice with HFD. The composition of the gut microbiota was altered with the exposure of cigarette smoking, and the change of the distribution of primary bile acids might be one of the reasons. It was concluded that cigarette smoking would break the homeostasis of cholesterol and bile acids metabolism and changed the composition of gut microbiota. Our discoveries confirmed that smoking bans are important for the public health.

Berberine improves liver injury induced glucose and lipid metabolic disorders via alleviating ER stress of hepatocytes and modulating gut microbiota in mice.

Liver injury mediated by endoplasmic reticulum (ER) stress can cause many kinds of liver diseases including hepatic glucose and lipid metabolic disorders, and long term liver injury would lead to cirrhosis and hepatic cancer. Therefore, effective drugs for treating liver injury are urgent in need. Berberine is a multifunctional drug of traditional Chinese medicine, and it can improve various liver diseases. To study the effects of berberine on ER stress-induced liver injury, tunicamycin was administrated to C57BL/6 mice with or without berberine pre-treatment. H&E staining was used to check the morphology and histology of liver tissues. The serum and liver tissues were harvested to test biochemical indexes and the expression levels of genes related with glucose and lipid metabolism, ER stress and unfold protein response (UPR). 16S rDNA sequence technology was conducted to check the fecal microbiota. Pre-administration with berberine could alleviate the excess accumulation of triglyceride (TG) in the liver of mice treated with tunicamycin. Tunicamycin administration caused significant increase of the expression level of genes related to ER stress and UPR, such as CHOP, Grp78 and ATF6, but the berberine pre-treatment could significantly downregulate the expression level of these genes. Tunicamycin administration resulted in increased ratio of Prevotellaceae to Erysipelotrichaceae at the family level of the fecal microbiota in mice, and this trend was reversed by the pre-treatment of berberine. These results demonstrated that berberine could improve liver injury induced hepatic metabolic disorders through relieving ER stress in hepatocytes and regulating gut microbiota in mice.

Effects of dihydroartemisinin on the gut microbiome of mice.

Dihydroartemisinin (DHA) is a semisynthetic derivative of artemisinin, which has been found to exhibit a broad range of biological activities, excluding antimalarial effects; however its effects on the gut microbiota remain poorly understood. The present study aimed to investigate the effects of DHA on the gut microbiome in mice and to determine its potential biological and pharmaceutical activities through its alteration of the gut microbiota. Serum glucose, triglyceride (TG), total cholesterol, lipopolysaccharide, high density lipoprotein­cholesterol, low density lipoprotein­cholesterol, alanine aminotransferase and aspartate aminotransferase levels in mice treated with DHA were analyzed using the corresponding detection kits. In addition, hematoxylin and eosin staining was performed to determine the pathological effects of DHA on the liver, kidney and intestinal tissues of mice, and the effects of DHA on the gut microbiome were analyzed using 16S ribosomal (r)DNA gene analysis. The results demonstrated that the TG serum levels of mice treated with DHA were significantly decreased compared with the control group. Furthermore, 16S rDNA gene analysis demonstrated that the bacterial diversity of mice treated with DHA was enriched compared with the control group. The DHA group exhibited increased numbers of Firmicutes and Saccharibacteria, and decreased Deferribacteres and Actinobacteria compared with the control group at the phylum level. Kyoto Encyclopedia of Genes and Genomes signaling pathway enrichment analysis also revealed that the signaling pathways associated with 'Energy metabolism' and 'Nucleotide metabolism' were upregulated, whereas the signaling pathways associated with 'Infectious diseases and 'Neurodegenerative diseases' were downregulated in the DHA group compared with the control group. In conclusion, the findings of the present study indicated that DHA may significantly decrease the serum TG levels and alter the gut microbiota, which suggested its potential to be used for the treatment of hyperlipidemia, inflammatory and neurodegenerative disorders.

Dihydroartemisinin ameliorates dextran sulfate sodium induced inflammatory bowel diseases in mice.

In the present study, the effects of dihydroartemisinin (DHA) on inflammatory bowel diseases (IBD) mice model induced by dextran sulfate sodium (DSS) were determined. Hematoxylin and eosin staining was used to assess the intestines of mice treated with DSS and DHA. The expression of inflammatory factors and cell junction-associated genes was measured using reverse transcription-quantitative PCR (RT-qPCR) and Western blot. The effects of DSS and DHA on the gut microbiome were measured using 16S recombinant (r) DNA gene analysis. DHA could improve the diarrhea and bloody stool induced by DSS, and decrease the serum levels of TNF-α, IL-1ß and IL-23 of the DSS group. DHA could notably reduce the infiltration of the inflammatory cells and significantly decrease the expression of TNF-α and IL-1ß in the intestines of the DSS treated mice. The expression of cell junction-associated genes such as EpCAM and Claudins, were down-regulated in the DSS group, and DHA could recover the expression of these cell junction-associated genes. The 16S rDNA gene analysis demonstrated that Bacteroidetes and Verrucomicrobia decreased, while Firmicutes and Proteobacteria increased in the DSS group, and DHA could recover the abundance of these gut bacteria altered by DSS. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that DHA could partly recover the pathways altered by DSS. DHA could obviously ameliorate the symptoms of IBD induced by DSS by regulation of the expression of inflammation and cell junction-associated genes and gut microbiota, suggesting its potential for the treatment of IBD.

Activation of Wnt/ß-catenin pathway causes insulin resistance and increases lipogenesis in HepG2 cells via regulation of endoplasmic reticulum stress.

BACKGROUND: Wnt/ß-catenin signaling is involved in glucose and lipid metabolism, but the mechanism is not clear yet. AIM: The objective is to study mechanisms of Wnt/ß-catenin signaling on regulating hepatocytes metabolism. METHODS: Real-time qPCR, Western blot, and Oil-red O staining methods were used. RESULTS: The Wnt/ß-catenin signaling was activated in hepatocytes by CP21R7, and the level of phosphorylated IRS-1 (Ser307) and TRB3 were significantly increased, while the levels of phosphorylated IRS-1 (Tyr612) and phosphorylated Akt were decreased. Moreover, the expression of FGF21, FAS, SCD1, PPARγ and ADRP was significantly increased. The expression of ATF4, ATF5, eIF2α, GRP78, CHOP and phosphorylated level of PERK were also increased. The expression of FGF21 and TRB3 was significantly down-regulated, and the lipid droplets were notably reduced after the ER stress was inhibited by TUDCA. The expression of FGF21 was significantly decreased when the IRE1 pathway of the UPR was inhibited by STF-083010. CONCLUSIONS: Activation of Wnt/ß-catenin signaling pathway could cause insulin resistance and lipogenesis in hepatocytes via regulation of the IRE1 pathway of the ER stress and UPR, providing new targets for the treatment of metabolic disorders.

Fatty liver and alteration of the gut microbiome induced by diallyl disulfide.

Diallyl disulfide (DADS) is one of the primary components of garlic and it exhibits a broad range of biological activities. In the present study, the effects of DADS on lipid metabolism and its potential role in the modulation of the gut microbiome were determined. Hematoxylin and eosin and oil­red O staining were used to assess the liver and intestinal tissues of mice treated with DADS. The expression of lipid metabolism­associated genes was measured using reverse transcription­quantitative PCR (RT­qPCR). The effects of DADS on the gut microbiome were measured using 16S recombinant (r)DNA gene analysis. The results revealed that the serum non­esterified free fatty acids, high density lipoprotein­cholesterol, low density lipoprotein­cholesterol, serum total cholesterol, liver triglyceride and total cholesterol levels of the mice fed with a low­dose of DADS was significantly higher when compared with the control. Hematoxylin and eosin and oil­red O staining demonstrated that DADS induced fatty liver in mice. The results of the RT­qPCR revealed that the expression levels of a number of lipid metabolism­associated genes were altered in the livers of mice treated with DADS. The 16S rDNA gene analysis demonstrated that the mice fed on a normal diet treated with a low­dose of DADS had decreased levels of bacteria from the Bacteroidetes phyla and increased levels of bacteria from the Firmicutes phyla. The Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed the top 20 pathways enriched in the low­dose DADS group of mice fed with a normal diet. In the present study, low­dose DADS induced fatty liver and altered the gut microbiota, similar to the phenotype induced by a high fat diet, by regulating the expression of lipid metabolism associated genes.

Circular RNA profile in liver tissue of EpCAM knockout mice.

Epithelial cell adhesion molecule (EpCAM) is highly expressed during liver development and carcinogenesis, However, its functions and underlying mechanisms remain unclear. Clustered regularly interspaced short palindromic repeats (CRISPRs)/CRISPR­associated protein 9 (Cas9) technology was used in the current study to establish EpCAM­/­ mice. The expression of EpCAM in the livers of the mice at embryonic day (E)18.5 and post­natal day (P)0 was detected by immunofluorescence staining. The expression of genes associated with the development and glycogen metabolism was also assessed by reverse transcription­quantitative PCR. Additionally, the liver tissue of the EpCAM­/­ and wild­type mice was used for non­coding RNA sequencing. The results of RNA sequencing revealed 11 up­regulated and 12 downregulated circular RNAs (circRNAs). Kyoto Encyclopedia of Genes and Genomes analysis for resource genes determined that the top altered pathways included cell junctions, cell cycle, immune signaling and metabolism. This analysis was also utilized to predict the target association of the circRNA­microRNA­mRNA network. The comprehensive liver tissue circRNA expression profiles produced in the present study may help to elucidate the functions and mechanisms of EpCAM during liver development.