ABSTRACT
Isoliquiritigenin (ISL) is an active chalcone compound isolated from licorice. It possesses anti-inflammatory and anti-oxidative activities. In our previous study, we uncovered a great potential of ISL in treatment of type 2 diabetes mellitus (T2DM). Therefore, this study aims to reveal the mechanism underlying the alleviatory effects of ISL on T2DM-induced glycolipid metabolism disorder. High-fat-high-sugar diet (HFD) combined with intraperitoneal injection of streptozotocin (STZ) were used to establish T2DM mice model. All animal experiments were carried out with approval of the Committee of Ethics at Beijing University of Chinese Medicine. HepG2 cells were used in in vitro experiments, and sodium palmitate (SP) was applied to establish insulin resistance (IR) model cells. The effects of ISL on body weight, fasting blood glucose levels, and pathological changes in the livers of mice were examined. Enzyme-linked immune sorbent assay (ELISA) and real-time quantitative PCR (RT-qPCR) were applied to detect the regulatory effects of ISL on key targets involved in glucolipid metabolism. Additionally, molecular docking and analytical dynamics simulation methods were used to analyze the interaction between ISL and key target protein. The results indicate that ISL significantly downregulates the transcriptional levels and inhibits the activities of key enzymes involved in gluconeogenesis, including pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), and fructose-1, 6-bisphosphatase (FBP). It also downregulates the transcriptional and protein levels of hepatocyte nuclear factor 4α (HNF4α) and cAMP response element binding protein (CREB), the two transcriptional factors involved in gluconeogenesis. Thus, ISL inhibits hepatic gluconeogenesis in T2DM mice. In addition, ISL reduces total cholesterol (TC) and triglyceride (TG) levels in the livers of T2DM mice. Moreover, ISL downregulates the mRNA levels of lipogenesis genes and upregulates those of genes involved in fatty acid oxidation, lipid uptake, and lipid export. In conclusion, ISL suppresses hepatic gluconeogenesis, promotes lipolysis, and restrains lipogenesis in T2DM mice, thereby improving the abnormal glycolipid metabolism caused by T2DM.
ABSTRACT
Isoliquiritigenin (ISL) is a flavonoid compound isolated from licorice. It possesses excellent antioxidant and anti-diabetic activities. This study aims to investigate the molecular mechanism underlying the alleviatory effect of ISL on energy metabolism imbalance caused by type 2 diabetes mellitus (T2DM). 8-week-old male C57BL/6J mice were used in in vivo experiments. The high-fat-high-glucose diet combined with intraperitoneal injection of streptozotocin was applied to establish T2DM animal model. All animal experiments were performed in accordance with the Institutional Guidelines of Laboratory Animal Administration issued by the Committee of Ethics at Beijing University of Chinese Medicine. HepG2 cells were used in in vitro experiments. Enzyme-linked immunosorbent assay (ELISA) and real-time quantitative polymerase chain reaction (RT-qPCR) were used to examine the protein and mRNA levels of mitochondrial function-related targets. The levels of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in HepG2 cells were measured by the flow cytometry. Additionally, the molecular docking of ISL and key target proteins was analyzed. It was found that ISL significantly inhibited the activity of mitochondrial respiratory chain complex I and increased the protein levels of uncoupling protein 2 (UCP2) in the livers of mice and HepG2 cells. It also obviously decreased the ROS levels and increased the MMP levels in cultured HepG2 cells. In addition, ISL promoted mitochondrial biogenesis by activating proliferator-activated receptor gamma co-activator 1α (PGC-1α) and enhanced mitophagy by upregulating Parkin. It also improved mitochondrial fusion by increasing the mRNA and protein levels of mitofusin 2 (MFN2). In conclusion, ISL alleviates energy metabolism imbalance caused by T2DM through suppression of excessive mitochondrial oxidative phosphorylation and promotion of mitochondrial biogenesis, mitophagy, and fusion.
ABSTRACT
1-Deoxy-D-xylulose-5-phosphate synthase (DXS) is a rate-limiting enzyme involved in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for terpenoid precursor biosynthesis. DXS plays an essential role in glycyrrhizic acid (GA) biosynthesis. Based on our previous transcriptome study, there was a negative correlation between DXS expression and GA content. Therefore, we explored the regulatory role of DXS in GA biosynthesis using both gene overexpression and gene knockout in a hairy root culture system. DXS was cloned from Glycyrrhiza glabra L. (GenBank Accession No. MN158121). A plant binary expression vector pCA-DXS was constructed by a gene fusion method. The sgRNA sequence was designed based on the first exon of DXS to construct the gene editing vector pHSE-DXS. Hairy roots overexpressing or knocking out DXS were generated through an Agrobacterium-mediated method with licorice hypocotyls as explants. Wild-type hairy roots and negative control hairy roots containing empty plasmids were also evaluated. UPLC was used to determine the GA content in each licorice hairy root line. Results showed that the content of GA in the hairy root group knocking out DXS was significantly higher than that in the wild-type and negative control groups, while in the hairy root group overexpressing DXS was significantly lower, suggesting that DXS plays a negative role in GA biosynthesis. This study provides a foundation for determining the function of DXS in terpenoid metabolism and for further establishment of a molecular regulatory network of GA biosynthesis.
ABSTRACT
Ferulate 5-hydroxylase (F5H) is a key enzyme involved in the phenylpropane metabolism pathway. Based on our previous transcriptome sequencing study, F5H played a negative regulatory role in glycyrrhizic acid (GA) biosynthesis. Therefore, in this study we cloned the F5H gene and investigated its regulatory effect on GA accumulation through gene overexpression and knockout. F5H was cloned from Glycyrrhiza glabra L. (GenBank Accession No. MK882511). A plant binary expression vector pCA-F5H was constructed by inserting F5H into pCAMBIA1305.1 at Spe I and Bgl II sites. The sgRNA sequences were designed based on the first exon of F5H. The CRISPR/Cas9 gene editing vector pHSE-F5H was constructed by inserting F5H sgRNA into pHSE401 at two Bsa Ⅰ sites. PCA-F5H and pHSE-F5H were transfected into Agrobacterium tumefaciens ATCC15834, which was used to induce hairy root overexpressing or knocking out F5H with licorice hypocotyl as explants. At the same time, wild type and negative control hairy roots were also generated. UPLC was used to assay the GA content in different hairy root lines, and results showed that the GA content in hairy root lines knocking out F5H was significantly higher, whereas in hairy root lines overexpressing F5H GA content was lower than that in the wild-type and negative control. In this work, through a reverse genetics strategy, the negative regulatory effect of F5H on GA biosynthesis was confirmed through gene overexpression and knockout. This work will lay a foundation for further elucidation of the molecular regulatory network of GA biosynthesis.
ABSTRACT
A simple, new aptamer-photonic crystal encoded suspension array was designed to simultaneously quantify and qualify ochratoxin A(OTA) and fumonisin B1(FB1) in cereal samples. The aptamers of OTA and FB1 were immobilized on the surfaces of photonic crystals by chemical bonding. When the target mycotoxins appear in a sample, the fluorescence-labeled complementary DNA of the aptamer dissociates from their double DNA hybrid and results in an obvious decrease in fluorescence intensity of the microsphere. The difference value of fluorescent intensities for each kind of silica photonic crystal microsphere (SPCM) quantitatively conveys the concentration of mycotoxin, and the structure colors or reflectance peak positions of the SPCMs confirm the kind of mycotoxin detected. The reaction conditions including the immobilization method for aptamers, hybridization, and incubation conditions have been optimized. This developed method displayed a wide linear detection range (0.01-1 ng/mL for OTA and 0.001-1 ng/mL for FB1) and a low limit of detection (0.25 pg/mL for OTA and 0.16 pg/mL for FB1). The recovery rates in the spiked cereal samples ranged from 81.80% to 116.38% for OTA and 76.58%-114.79% for FB1. The positive detection results in the naturally contaminated cereal samples were in agreement with those of classic enzyme-linked immunosorbent assay (ELISA). This simple suspension array scheme displays a great application potential for the high throughput screen assay of mycotoxins.
Subject(s)
Aptamers, Nucleotide/chemistry , Edible Grain/chemistry , Fumonisins/analysis , Ochratoxins/analysis , Microspheres , Photons , Silicon Dioxide/chemistryABSTRACT
Bikunin is a proteoglycan exhibiting broad-spectrum inhibitory activity against serine proteases and could potentially suppress tumor cell invasion and metastasis. Here, we have successfully expressed recombinant human bikunin (rh-bikunin) in the methylotrophic yeast Pichia pastoris and established the purification procedure. The cDNA encoding human bikunin was cloned by PCR and inserted into the expression vector pPICZalphaC. After expressed in shake flask, rh-bikunin was produced in an 80-L fermenter and purified by cation exchange chromatography and reverse phase chromatography. The rh-bikunin was active by trypsin inhibition test. The final expression levels were 55 mg/L and we got totally 1.44 g (5600 inhibitor units/mg) of purified rh-bikunin (purity is 95%) from 40 L of fermentation broth. The rh-bikunin consists of two forms with molecular masses of 24 and 21 kDa, respectively. Both forms were immunoreactive by Western blotting and N-terminals were correctly processed by amino-terminal sequencing. This study provided a new method for expression and purification of active rh-bikunin.
