Hypothalamic POMC neuron-specific knockout of MC4R affects insulin sensitivity by regulating Kir2.1.

BACKGROUND: Imbalance in energy regulation is a major cause of insulin resistance and diabetes. Melanocortin-4 receptor (MC4R) signaling at specific sites in the central nervous system has synergistic but non-overlapping functions. However, the mechanism by which MC4R in the arcuate nucleus (ARC) region regulates energy balance and insulin resistance remains unclear. METHODS: The MC4Rflox/flox mice with proopiomelanocortin (POMC) -Cre mice were crossed to generate the POMC-MC4Rflox/+ mice. Then POMC-MC4Rflox/+ mice were further mated with MC4Rflox/flox mice to generate the POMC-MC4Rflox/flox mice in which MC4R is selectively deleted in POMC neurons. Bilateral injections of 200 nl of AAV-sh-Kir2.1 (AAV-sh-NC was used as control) were made into the ARC of the hypothalamus. Oxygen consumption, carbon dioxide production, respiratory exchange ratio and energy expenditure were measured by using the CLAMS; Total, visceral and subcutaneous fat was analyzed using micro-CT. Co-immunoprecipitation assays (Co-IP) were used to analyze the interaction between MC4R and Kir2.1 in GT1-7 cells. RESULTS: POMC neuron-specific ablation of MC4R in the ARC region promoted food intake, impaired energy expenditure, leading to increased weight gain and impaired systemic glucose homeostasis. Additionally, MC4R ablation reduced the activation of POMC neuron, and is not tissue-specific for peripheral regulation, suggesting the importance of its central regulation. Mechanistically, sequencing analysis and Co-IP assay demonstrated a direct interaction of MC4R with Kir2.1. Knockdown of Kir2.1 in POMC neuron-specific ablation of MC4R restored the effect of MC4R ablation on energy expenditure and systemic glucose homeostasis, indicating by reduced body weight and ameliorated insulin resistance. CONCLUSION: Hypothalamic POMC neuron-specific knockout of MC4R affects energy balance and insulin sensitivity by regulating Kir2.1. Kir2.1 represents a new target and pathway that could be targeted in obesity.

Modulating the polarization phenotype of microglia - A valuable strategy for central nervous system diseases.

Central nervous system (CNS) diseases have become one of the leading causes of death in the global population. The pathogenesis of CNS diseases is complicated, so it is important to find the patterns of the disease to improve the treatment strategy. Microglia are considered to be a double-edged sword, playing both harmful and beneficial roles in CNS diseases. Therefore, it is crucial to understand the progression of the disease and the changes in the polar phenotype of microglia to provide guidance in the treatment of CNS diseases. Microglia activation may evolve into different phenotypes: M1 and M2 types. We focused on the roles that M1 and M2 microglia play in regulating intercellular dialogues, pathological reactions and specific diseases in CNS diseases. Importantly, we summarized the strategies used to modulate the polarization phenotype of microglia, including traditional pharmacological modulation, biological therapies, and physical strategies. This review will contribute to the development of potential strategies to modulate microglia polarization phenotypes and provide new alternative therapies for CNS diseases.

Zuo Gui Wan Promotes Osteogenesis via PI3K/AKT Signaling Pathway: Network Pharmacology Analysis and Experimental Validation.

OBJECTIVE: Osteogenesis is vitally important for bone defect repair, and Zuo Gui Wan (ZGW) is a classic prescription in traditional Chinese medicine (TCM) for strengthening bones. However, the specific mechanism by which ZGW regulates osteogenesis is still unclear. The current study is based on a network pharmacology analysis to explore the potential mechanism of ZGW in promoting osteogenesis. METHODS: A network pharmacology analysis followed by experimental validation was applied to explore the potential mechanisms of ZGW in promoting the osteogenesis of bone marrow mesenchymal stem cells (BMSCs). RESULTS: In total, 487 no-repeat targets corresponding to the bioactive components of ZGW were screened, and 175 target genes in the intersection of ZGW and osteogenesis were obtained. And 28 core target genes were then obtained from a PPI network analysis. A GO functional enrichment analysis showed that the relevant biological processes mainly involve the cellular response to chemical stress, metal ions, and lipopolysaccharide. Additionally, KEGG pathway enrichment analysis revealed that multiple signaling pathways, including the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway, were associated with ZGW-promoted osteogensis. Further experimental validation showed that ZGW could increase alkaline phosphatase (ALP) activity as well as the mRNA and protein levels of ALP, osteocalcin (OCN), and runt related transcription factor 2 (Runx 2). What's more, Western blot analysis results showed that ZGW significantly increased the protein levels of p-PI3K and p-AKT, and the increases of these protein levels significantly receded after the addition of the PI3K inhibitor LY294002. Finally, the upregulated osteogenic-related indicators were also suppressed by the addition of LY294002. CONCLUSION: ZGW promotes the osteogenesis of BMSCs via PI3K/AKT signaling pathway.

Integrated Network Pharmacology and Gut Microbiota Analysis to Explore the Mechanism of Sijunzi Decoction Involved in Alleviating Airway Inflammation in a Mouse Model of Asthma.

Background: Asthma is a chronic inflammatory disease of the airways with recurrent attacks, which seriously affects the patients' quality of life and even threatens their lives. The disease can even threaten the lives of patients. Sijunzi decoction (SJZD), a classical Chinese medicine formula with a long history of administration, is a basic formula used for the treatment of asthma and demonstrates remarkable efficacy. However, the underlying mechanism has not been elucidated. Materials and Methods: We aimed to integrate network pharmacology and intestinal flora sequencing analysis to study the mechanism of SJZD in the treatment of allergic asthmatic mice. The active compounds of SJZD and their asthma-related targets were predicted by various databases. We performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses to identify potentially relevant pathways for target genes. Furthermore, the active compound-target and target-signaling pathway network maps were constructed by using Cytoscape 3.8.2. These results were combined with those of the intestinal flora sequencing analysis to study the influence of SJZD on airway inflammation in allergic asthmatic mice. Result: We obtained 137 active compounds from SJZD and associated them with 1445 asthma-related targets acquired from the databases. A total of 109 common targets were identified. We visualized active compound-target and target-signaling pathway network maps. The pathological analysis and inflammation score results suggested that SJZD could alleviate airway inflammation in asthmatic mice. Sequencing analysis of intestinal flora showed that SJZD could increase the relevant abundance of beneficial bacterial genus and maintain the balance of the intestinal flora. The core toll-like receptor (TLR) signaling pathway was identified based on network pharmacology analysis, and the important role TLRs play in intestinal flora and organismal immunity was also recognized. The analysis of the correlation between environmental factors and intestinal flora revealed that beneficial bacterial genera were negatively correlated with TLR2 and positively correlated with the TLR7 expression. Furthermore, they were positively correlated with IFN-γ and IL-10 levels and negatively correlated with IL-4 and IL-17 levels. Conclusion: SJZD alleviated the airway inflammation state in asthmatic mice. The findings suggest that increasing the relevant abundance of beneficial intestinal bacteria in mice with asthma, regulating intestinal flora, interfering with the level of TLR2 and TLR7 expression to adjust the secretion of inflammatory factors, and alleviating asthmatic airway inflammation may be the possible mechanism involved in the treatment of asthma by SJZD, providing a basis for further studies on SJZD.

Effects of sweeteners on host physiology by intestinal mucosal microbiota: Example-addition sweeteners in Qiweibaizhu Powder on intestinal mucosal microbiota of mice with antibiotic-associated diarrhea.

In recent years, sweeteners have gained massive popularity under the trend of limiting sugar intake. Our previous study found that Qiweibaizhu Powder (QWBZP) could improve gut microbiota dysbiosis and has good efficacy in treating antibiotic-associated diarrhea (AAD). In this study, we investigated the effects of sucrose, sorbitol, xylitol, and saccharin on the intestinal mucosal microbiota of AAD mice treated with QWBZP. When the AAD model was constructed by being gavaged mixed antibiotic solution, Kunming mice were randomly assigned to seven groups: the control (mn) group, the ADD (mm) group, the QWBZP (mq) group, the saccharin + QWBZP (mc) group, the sucrose + QWBZP (ms) group, the xylito + QWBZP (mx) group, and the sorbitol + QWBZP (msl) group. Subsequently, 16S rRNA gene amplicon sequencing was used to analyze the intestinal mucosal microbiota composition and abundance. The results showed that feces from AAD mice were diluted and wet and improved diarrhea symptoms with QWBZP and sorbitol. In contrast, the addition of sucrose, saccharin, and xylitol delayed the healing of diarrhea. The relative abundance of intestinal mucosal microbiota showed Glutamicibacter, Robinsoniella, and Blautia were characteristic bacteria of the mx group, Candidatus Arthromitus, and Bacteroidales_S24-7_group as the typical bacteria of the mn group, Clostridium_innocuum_group as the distinct bacteria of the mm group. Mycoplasma and Bifidobacterium as the characteristic bacteria of the ms group. Correlation analysis of typical bacterial genera with metabolic functions shows that Blautia negatively correlates with D-Glutamine and D-glutamate metabolism. Bacteroidales_S24-7_group has a significant negative correlation with the Synthesis and degradation of ketone bodies. The study confirmed that sucrose, sorbitol, xylitol, and saccharin might further influence metabolic function by altering the intestinal mucosal microbiota. Compared to the other sweetener, adding sorbitol to QWBZP was the best therapeutic effect for AAD and increased the biosynthesis and degradation activities. It provides the experimental basis for applying artificial sweeteners in traditional Chinese medicine (TCM) as a reference for further rational development and safe use of artificial sweeteners.

Comparative Analysis of the Gut Microbiota in Mice under Lard or Vegetable Blend Oil Diet.

Diet is the most direct and rapid contributor to the gut microbiome. Oils and fats are important nutrients in the human body. The effects of lard or vegetable blend oil on gut microbiota were investigated. Kunming mice were given lard or vegetable blend oil for six weeks. Changes in microbiota composition and abundance in lard or vegetable blend oil diets were analyzed by 16S rRNA gene amplicon sequencing. Our study shows that the gut microbiota of mice changed significantly after ingestion of lard or vegetable blend oil. Lard may synergize with Coriobacteriaceae_UCG-002. Vegetable blend oil has synergistic effects with Akkermansia, Roseburia, and Enteractinococcus. Coriobacteriaceae_UCG-002 showed a significant negative correlation with Glycolysis/Gluconeogenesis. Roseburia was most strongly associated with Starch and sucrose metabolism. According to bacterial function prediction and correlation analysis, long-term consumption of lard or vegetable oil may affect glycolipid metabolism, but lard has a greater impact on human health and consequently host health.

Gut-Kidney Impairment Process of Adenine Combined with Folium sennae-Induced Diarrhea: Association with Interactions between Lactobacillus intestinalis, Bacteroides acidifaciens and Acetic Acid, Inflammation, and Kidney Function.

BACKGROUND: Extensive evidence suggests that gut microbiota may interact with the kidneys and play central roles in the pathogenesis of disease. However, the association of gut microbiota-kidneys in diarrhea remains unclear. METHODS: A diarrhea mouse model was constructed by combining adenine with Folium sennae. We analyzed the characteristics of the gut content microbiota and short chain fatty acids (SCFAs); and explored the potential link between gut content microbiota, SCFAs, intestinal inflammatory response and kidney function. RESULTS: Characteristic bacteria Lactobacillus intestinalis and Bacteroides acidifaciens were enriched in the gut contents of mice. The productions of SCFAs were remarkably inhibited. Model mice presented an increased trend of creatinine (Cr), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), a decreased trend of blood urea nitrogen (BUN) and secretory immunoglobulin A (SIgA). The pathological analysis proved obvious damage to the kidney structure. Lactobacillus intestinalis and Bacteroides acidifaciens exisited in the correlations with acetic acid, intestinal inflammatory response and kidney function. CONCLUSIONS: Adenine combined with Folium sennae-induced diarrhea, altered the structure and function of the gut content microbiota in mice, causing the enrichment of the characteristic bacteria Lactobacillus intestinalis and Bacteroides acidifaciens. The interactions between Lactobacillus intestinalis, Bacteroides acidifaciens and acetic acid, intestinal inflammation, and kidney function might be involved in the process of gut-kidney impairment in adenine, combined with Folium sennae-induced diarrhea.

Importance of Dendrobium officinale in improving the adverse effects of high-fat diet on mice associated with intestinal contents microbiota.

A growing body of evidence suggests that the disturbance of intestinal microbiota induced by high-fat diet is the main factor causing many diseases. Dendrobium officinale (DO), a medicinal and edible homologous Chinese herbal medicine, plays essential role in regulating intestinal microbiota. However, the extent of DO on the intestinal contents microbiota in mice fed with a high-fat diet still remains unclear. Therefore, this study explored the role of intestinal contents microbiota in the regulation of adverse effects caused by high-fat diet by DO from the perspective of intestinal microecology. Twenty-four mice were randomly distributed into the normal saline-treated basal diet (bcn), normal saline-treated high-fat diet (bmn), 2.37 g kg-1 days-1 DO traditional decoction-treated high-fat diet (bdn) and 1.19 g kg-1 days-1 lipid-lowering decoction-treated high-fat diet (bjn) groups for 40 days. Subsequently, we assessed the changes in body weight, serum total cholesterol (TC), total triacylglycerol (TG), low density lipoprotein-cholesterol (LDL-C), high density lipoprotein-cholesterol (HDL-C) levels, and the characteristics of intestinal contents microbiota. Results demonstrated that DO exerted the modulating effect on the changes in body weight, TG, TC, LDL-C, and HDL-C levels. Besides, DO decreased the richness and diversity of intestinal contents microbiota, and altered the structure as a whole. Dominant bacteria, Ruminococcus and Oscillospira, varied significantly and statistically. Moreover, DO influenced the carbohydrate, amino acid, and energy metabolic functions. Furthermore, Ruminococcus and Oscillospira presented varying degrees of inhibition/promotion of TG, TC, LDL-C, and HDL-C. Consequently, we hypothesized that Ruminococcus and Oscillospira, as dominant bacteria, played key roles in the treatment of diseases associated with a high-fat diet DO.

Renoprotective effects of ferric citrate in a mouse model of chronic kidney disease.

In chronic kidney disease, ferric citrate has been shown to be an effective phosphate binder and source of enteral iron; however, the effects of ferric citrate on the kidney have been less well-studied. Here, in Col4α3 knockout mice-a murine model of progressive chronic kidney disease, we evaluated the effects of five weeks of 1% ferric citrate dietary supplementation. As expected, ferric citrate lowered serum phosphate concentrations and increased serum iron levels in the Col4α3 knockout mice. Consistent with decreased enteral phosphate absorption and possibly improved iron status, ferric citrate greatly reduced circulating fibroblast growth factor 23 levels. Interestingly, ferric citrate also lessened systemic inflammation, improved kidney function, reduced albuminuria, and decreased kidney inflammation and fibrosis, suggesting renoprotective effects of ferric citrate in the setting of chronic kidney disease. The factors mediating possible ferric citrate renoprotection, the mechanisms by which they may act, and whether ferric citrate affects chronic kidney disease progression in humans deserves further study.

Different Effects of Lard and Vegetable Blend Oil on Intestinal Microorganisms, Enzyme Activity and Blood Routine in Mice.

The intake of moderate oils and fats is necessary to maintain the body's energy balance, and the fatty acid composition of different oils and fats varies in their nutrition and function. The study aimed to investigate the effects of lard and vegetable blend oil on gut microbiota, intestinal enzyme activities, and blood routine. Kunming mice were assigned to the three groups: (1) Control group (CK) was gavage administration with distilled water, (2) Plant oil group (ZWY) was gavage administration with edible vegetable blend oil, (3) Lard group (DWY) was gavage administration with lard. After 42 days, microbiological, digestive enzymes, and blood routine were performed. Compared with the CK group, Escherichia coli, Lactobacilli, and Bifidobacteria were significantly decreased (p < 0.05), the activities of protease, cellulase, amylase, and xylanase were markedly reduced (p < 0.05), the hemoglobin was significantly increased (p < 0.05) in the ZWY group and DWY groups, and the hematocrit was increased in the ZWY group (p < 0.05), while other routine blood indices were increased (p > 0.05). Compared to the ZWY group, the activity of cellulase and amylase were significantly increased (p < 0.05), the intestinal microorganism and the routine blood indexes had no significant difference in the DWY group. Lard and vegetable blend oil diet affected the composition of the intestinal microorganisms, and the functions of digestive enzymes. Meanwhile, the levels of digestive enzymes may be correlated with the intestinal microbiota.

Enteral ferric citrate absorption is dependent on the iron transport protein ferroportin.

Ferric citrate is approved as an iron replacement product in patients with non-dialysis chronic kidney disease and iron deficiency anemia. Ferric citrate-delivered iron is enterally absorbed, but the specific mechanisms involved have not been evaluated, including the possibilities of conventional, transcellular ferroportin-mediated absorption and/or citrate-mediated paracellular absorption. Here, we first demonstrate the efficacy of ferric citrate in high hepcidin models, including Tmprss6 knockout mice (characterized by iron-refractory iron deficiency anemia) with and without adenine diet-induced chronic kidney disease. Next, to assess whether or not enteral ferric citrate absorption is dependent on ferroportin, we evaluated the effects of ferric citrate in a tamoxifen-inducible, enterocyte-specific ferroportin knockout murine model (Villin-Cre-ERT2, Fpnflox/flox). In this model, ferroportin deletion was efficient, as tamoxifen injection induced a 4000-fold decrease in duodenum ferroportin mRNA expression, with undetectable ferroportin protein on Western blot of duodenal enterocytes, resulting in a severe iron deficiency anemia phenotype. In ferroportin-deficient mice, three weeks of 1% ferric citrate dietary supplementation, a dose that prevented iron deficiency in control mice, did not improve iron status or rescue the iron deficiency anemia phenotype. We repeated the conditional ferroportin knockout experiment in the setting of uremia, using an adenine nephropathy model, where three weeks of 1% ferric citrate dietary supplementation again failed to improve iron status or rescue the iron deficiency anemia phenotype. Thus, our data suggest that enteral ferric citrate absorption is dependent on conventional enterocyte iron transport by ferroportin and that, in these models, significant paracellular absorption does not occur.

A Network Pharmacology Approach to Explore the Potential Mechanisms of Yifei Sanjie Formula in Treating Pulmonary Fibrosis.

OBJECTIVE: Yifei Sanjie Formula (YFSJF) is an effective formula on pulmonary fibrosis (PF), which has been used in clinic for more than 30 years. In order to investigate the molecular mechanism of YFSJF in treating PF, network pharmacology was used to predict the cooperative ingredients and associated pathways. METHODS: Firstly, we collected potential active ingredients of YFSJF by TCMSP databases. Secondly, we obtained PF-associated targets through OMIM and Genecards database. Finally, metascape was applied for the analysis of GO terms and KEGG pathways. RESULTS: We screened out 76 potential active ingredients and 98 associated proteins. A total of 5715 items were obtained by GO enrichment analysis (P < 0.05), including 4632 biological processes, 444 cellular components, and 639 molecular functions. A total of 143 related KEGG pathways were enriched (P < 0.05), including IL-17 signaling pathway, T cell receptor signaling pathway, TNF signaling pathway, calcium signaling pathway, TH17 cell differentiation, HIF-1 signaling pathway, and PI3K-Akt signaling pathway. CONCLUSION: YFSJF can interfere with immune and inflammatory response through multiple targets and pathways, which has a certain role in the treatment of PF. This study lays a foundation for future experimental research.

Minihepcidins are rationally designed small peptides that mimic hepcidin activity in mice and may be useful for the treatment of iron overload.

Iron overload is the hallmark of hereditary hemochromatosis and a complication of iron-loading anemias such as ß-thalassemia. Treatment can be burdensome and have significant side effects, and new therapeutic options are needed. Iron overload in hereditary hemochromatosis and ß-thalassemia intermedia is caused by hepcidin deficiency. Although transgenic hepcidin replacement in mouse models of these diseases prevents iron overload or decreases its potential toxicity, natural hepcidin is prohibitively expensive for human application and has unfavorable pharmacologic properties. Here, we report the rational design of hepcidin agonists based on the mutagenesis of hepcidin and the hepcidin-binding region of ferroportin and computer modeling of their docking. We identified specific hydrophobic/aromatic residues required for hepcidin-ferroportin binding and obtained evidence in vitro that a thiol-disulfide interaction between ferroportin C326 and the hepcidin disulfide cage may stabilize binding. Guided by this model, we showed that 7­9 N-terminal amino acids of hepcidin, including a single thiol cysteine, comprised the minimal structure that retained hepcidin activity, as shown by the induction of ferroportin degradation in reporter cells. Further modifications to increase resistance to proteolysis and oral bioavailability yielded minihepcidins that, after parenteral or oral administration to mice, lowered serum iron levels comparably to those after parenteral native hepcidin. Moreover, liver iron concentrations were lower in mice chronically treated with minihepcidins than those in mice treated with solvent alone. Minihepcidins may be useful for the treatment of iron overload disorders.