[Evaluation of chemical constituents of Draconis Sanguis and its protective effect on renal tubular injury in diabetic kidney disease].

The chemical constituents of Draconis Sanguis were preliminarily studied by macroporous resin, silica gel, dextran gel, and high-performance liquid chromatography. One retro-dihydrochalcone, four flavonoids, and one stilbene were isolated. Their chemical structures were identified as 4-hydroxy-2,6-dimethoxy-3-methyldihydrochalcone(1), 4'-hydroxy-5,7-dimethoxy-8-methylflavan(2), 7-hydroxy-4',5-dimethoxyflavan(3),(2S)-7-hydroxy-5-methoxy-6-methylflavan(4),(2S)-7-hydroxy-5-methoxyflavan(5), and pterostilbene(6) by modern spectroscopy, physicochemical properties, and literature comparison. Compound 1 was a new compound. Compounds 2 and 6 were first found in the Arecaceae family. Compound 5 had the potential to prevent and treat diabetic kidney disease.

[Effect and mechanism of Zexie Decoction in promoting white adipose tissue browning/brown adipose tissue activation based on GLP-1R/cAMP/PKA/CREB pathway].

This study investigated the mechanism of Zexie Decoction(ZXD) in promoting white adipose tissue browning/brown adipose tissue activation based on the GLP-1R/cAMP/PKA/CREB pathway. A hyperlipidemia model was induced by a western diet(WD) in mice, and the mice were divided into a control group, a model group(WD), and low-, medium-, and high-dose ZXD groups. An adipogenesis model was induced in 3T3-L1 cells in vitro, and with forskolin(FSK) used as a positive control, low-, medium-, and high-dose ZXD groups were set up. Immunohistochemistry and immunofluorescence results showed that compared with the WD group, ZXD promoted the expression of UCP1 in white and brown adipose tissues, and also upregulated UCP1, CPT1ß, PPARα, and other genes in the cells. Western blot analysis showed a dose-dependent increase in the protein expression of PGC-1α, UCP1, and PPARα with ZXD treatment, indicating that ZXD could promote the white adipose tissue browning/brown adipose tissue activation. Hematoxylin-eosin(HE) staining results showed that after ZXD treatment, white and brown adipocytes were significantly reduced in size, and the mRNA expression of ATGL, HSL, MGL, and PLIN1 was significantly upregulated as compared with the results in the WD group. Oil red O staining and biochemical assays indicated that ZXD improved lipid accumulation and promoted lipolysis. Immunohistochemistry and immunofluorescence staining for p-CREB revealed that ZXD reversed the decreased expression of p-CREB caused by WD. In vitro intervention with ZXD increased the protein expression of CREB, p-CREB, and p-PKA substrate, and increased the mRNA level of CREB. ELISA detected an increase in intracellular cAMP concentration with ZXD treatment. Molecular docking analysis showed that multiple active components in Alismatis Rhizoma and Atractylodis Macrocephalae Rhizoma could form stable hydrogen bond interactions with GLP-1R. In conclusion, ZXD promotes white adipose tissue browning/brown adipose tissue activation both in vivo and in vitro, and its mechanism of action may be related to the GLP-1R/cAMP/PKA/CREB pathway.

[Zexie Decoction regulates Akt/TFEB signaling pathway to promote lipophagy in hepatocytes].

Taking lipophagy as the breakthrough point, we explored the mechanism of Zexie Decoction(ZXD) in improving lipid metabolism in the hepatocyte model induced by palmitic acid(PA) and in the animal model induced by high-fat diet(HFD) on the basis of protein kinase B(Akt)/transcription factor EB(TFEB) signaling pathway. Co-localization was carried out for the microtubule-associated protein light chain 3(LC3) plasmid labeled with green fluorescent protein(GFP) and lipid droplets(LDs), and immunofluorescence co-localization for liver LC3 of HFD mice and perilipin 2(PLIN2). The results showed that ZXD up-regulated the expression of LC3, reduced lipid accumulation in hepatocytes, and increased the co-localization of LC3 and LDs, thereby activating lipo-phagy. Western blot results confirmed that ZXD increased autophagy-related protein LC3Ⅱ/LC3Ⅰ transformation ratio and lysosome-associated membrane protein 2(LAMP2) in vivo and in vitro and promoted the degradation of sequestosome-1(SQSTM1/p62)(P<0.05). The results above jointly explained that ZXD regulated lipophagy. Furthermore, ZXD activated TFEB expression(P<0.05) and reversed the PA-and HFD-induced decrease of TFEB nuclear localization in hepatocytes(P<0.05). Meanwhile, ZXD activated liver TFEB to up-regulate the expression of the targets Lamp2, Lc3 B, Bcl2, and Atg5(P<0.05). Additionally, ZXD down-regulated the protein level of p-Akt upstream of TFEB in vivo and in vitro. In conclusion, ZXD may promote lipophagy by regulating the Akt/TFEB pathway.

Sesquiterpene Lactams and Lactones With Antioxidant Potentials From Atractylodes macrocephala Discovered by Molecular Networking Strategy.

Atractylodes macrocephala rhizome (called Bái-zhú in China) has a long history as a functional food and herbal medicine in East Asia, especially China. Sesquiterpenoids are one of the main active compounds of Atractylodes macrocephala rhizome. This study aimed to explore the unknown sesquiterpenoids of A. macrocephala rhizome using a molecular networking strategy. Two new nitrogen-containing sesquiterpenoids, atractylenolactam A (1) and atractylenolactam B (2), and 2 new sesquiterpene lactones, 8-methoxy-atractylenolide V (6) and 15-acetoxyl atractylenolide III (7), along with 12 known analogs (3-5 and 8-16) were discovered and isolated. All the structures were assigned based on detailed spectroscopic analyses. The absolute configurations of 1, 2, 6, and 7 were established by time-dependent density functional theory ECD (TDDFT-ECD) calculations. All these compounds had different degrees of concentration-dependent activating effects on nuclear-factor-E2-related factor-2 (Nrf2).

[New eudesmane sesquiterpenoids from Atractylodis Macrocephalae Rhizoma and their inhibitory activities against SREBPs].

Three sesquiterpenoids were isolated and purified from the 95% ethanol extract of Atractylodis Macrocephalae Rhizoma by column chromatography on silica gel, Sephadex LH-20, ODS, and high-performance liquid chromatography(HPLC). Their chemical structures were identified on the basis of spectroscopic analysis and physiochemical properties as(7Z)-8ß,13-diacetoxy-eudesma-4(15),7(11)-diene(1), 7-oxo-7,8-secoeudesma-4(15),11-dien-8-oic acid(2), and guai-10(14)-en-11-ol(3). Compounds 1 and 2 are new compounds and compound 3 was obtained from Compositae family for the first time. Compounds 1, 2, and 3 showed weak inhibitory activities against sterol regulatory element-binding proteins(SREBPs).

[Mechanism of Zexie Decoction in improvement of nonalcoholic fatty liver disease based on LKB1/AMPK/PGC-1α pathway].

The present study investigated the pharmaceutical effect and underlying mechanism of Zexie Decoction(ZXD) on nonalcoholic fatty liver disease(NAFLD) in vitro and in vivo via the LKB1/AMPK/PGC-1α pathway based on palmitic acid(PA)-induced lipid accumulation model and high-fat diet(HFD)-induced NAFLD model in mice. As revealed by the MTT assay, ZXD had no effect on HepG2 activity, but dose-dependently down-regulated alanine aminotransferase(ALT) and aspartate aminotransferase(AST) in the liver cell medium induced by PA, and decreased the plasma levels of ALT and AST, and total cholesterol(TC) and triglyceride(TG) levels in the liver. Nile red staining showed PA-induced intracellular lipid accumulation, significantly increased lipid accumulation of hepatocytes induced by PA, suggesting that the lipid accumulation model in vitro was properly induced. ZXD could effectively improve the lipid accumulation of hepatocytes induced by PA. Oil red O staining also demonstrated that ZXD improved the lipid accumulation in the liver of HFD mice. JC-1 staining for mitochondrial membrane potential indicated that ZXD effectively reversed the decrease in mitochondrial membrane potential caused by hepatocyte injury induced by PA, activated PGC-1α, and up-regulated the expression of its target genes, such as ACADS, CPT-1α, CPT-1ß, UCP-1, ACSL-1, and NRF-1. In addition, as revealed by the Western blot and immunohistochemistry, ZXD up-regulated the protein expression levels of LKB1, p-AMPK, p-ACC, and PGC-1α in vivo and in vitro. In conclusion, ZXD can improve NAFLD and its mechanism may be related to the regulation of the LKB1/AMPK/PGC-1α pathway.

Two new sesquiterpenes from the rhizomes of Atractylodes macrocephala and their biological activities.

Two new sesquiterpenes, named selina-4(14),7,11-trien-9-ol (1) and selina-4(14),11-dien-7-ol (2), along with two known compounds were isolated from rhizomes of Atractylodes macrocephala Koidz. All structures were assigned on the basis of detailed spectroscopic analyses. The absolute configuration of 1 was established by TDDFT-ECD calculations. Compound 1 was found to moderately inhibit LSD1 activity with IC50 value of 34.0 µM. Compounds 1 and 4 exhibited a regulate effect on Keap1-Nrf2-ARE pathway.[Formula: see text].

Four new sesquiterpene lactones from Atractylodes macrocephala and their CREB agonistic activities.

One new bisesquiterpenoid, biepiasreorlid II (1), three new sesquiterpene lactones 8α-methoxy-epiasterolid (4), 3ß-acetoxyl-8-epiasterolid (5), and 3ß-acetoxyl-atractylenolide I (6), along with five known analogues (2-3 and 7-9), were obtained from rhizome of Atractylodes macrocephala Koidz. All structures were assigned on the basis of detailed spectroscopic analyses. The absolute configuration of 1 was established by the analysis of single-crystal X-ray diffraction with Ga Kα radiation, and 4-6 were elucidated by TDDFT-ECD calculations. The CREB agonistic activity was investigated in HEK293T cells using dual luciferase reporter assay. Compounds 1, 2, 5, and 7-9 exhibited strong to agonistic activities on CREB.

Isoproturon-Induced Salicylic Acid Confers Arabidopsis Resistance to Isoproturon Phytotoxicity and Degradation in Plants.

This study identified the effect of salicylic acid on degradation of isoproturon in Arabidopsis. Three T-DNA insertion mutant lines pal1- 1, pal1- 2, and eps1- 1 defective in salicylic acid synthesis were tested, which showed higher isoproturon accumulation and a toxic symptom in the mutants. When treated with 5 mg/L salicylic acid, these lines displayed a lower level of isoproturon and showed an attenuated toxic symptom. An RNA-sequencing study identified 2651 (1421 up and 1230 down) differentially expressed genes (DEGs) in eps1- 1 and 2211 (1556 up and 655 down) in pal1- 2 mutant plants (>2.0 fold change, p < 0.05). Some of the DEGs covered Phase I-III reaction components, like glycosyltransferases (GTs) and ATP-binding cassette transporters (ABCs). Using ultra performance liquid chromatography-time-of-flight-tandem-mass spectrometer/mass spectrometer (UPLC/Q-TOF-MS/MS), 13 Phase I and four Phase II metabolites were characterized. Of these, two metabolites 1-OH-isopropyl-benzene-O-glucoside and 4-isopropylphenol-S-2-methylbutanoyl-serine, have been identified and reported for the first time.

Detoxification of Atrazine by Low Molecular Weight Thiols in Alfalfa (Medicago sativa).

Low molecular weight (LMW) thiols in higher plants are a group of sulfur-rich nonprotein compounds and play primary and multiple roles in cellular redox homeostasis, enzyme activities, and xenobiotics detoxification. This study focused on identifying thiols-related protein genes from the legume alfalfa exposed to the herbicide atrazine (ATZ) residues in environment. Using high-throughput RNA-sequencing, a set of ATZ-responsive thiols-related protein genes highly up-regulated and differentially expressed in alfalfa was identified. Most of the differentially expressed genes (DEGs) were involved in regulation of biotic and abiotic stress responses. By analyzing the genes involved in thiols-mediated redox homeostasis, we found that many of them were thiols-synthetic enzymes such as γ-glutamylcysteine synthase (γECS), homoglutathione synthetase (hGSHS), and glutathione synthetase (GSHS). Using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), we further characterized a group of ATZ-thiols conjugates, which are the detoxified forms of ATZ in plants. Cysteine S-conjugate ATZ-HCl+Cys was the most important metabolite detected by MS. Several other ATZ-conjugates were also examined as ATZ-detoxified metabolites. Such results were validated by characterizing their analogs in rice. Our data showed that some conjugates under ATZ stress were detected in both plants, indicating that some detoxified mechanisms and pathways can be shared by the two plant species. Overall, these results indicate that LMW thiols play critical roles in detoxification of ATZ in the plants.

Degrading and Phytoextracting Atrazine Residues in Rice (Oryza sativa) and Growth Media Intensified by a Phase II Mechanism Modulator.

Atrazine (ATZ) residue in farmland is one of the environmental contaminants seriously affecting crop production and food safety. Understanding the regulatory mechanism for ATZ metabolism and degradation in plants is important to help reduce ATZ potential toxicity to both plants and human health. Here, we report our newly developed engineered rice overexpressing a novel Phase II metabolic enzyme glycosyltransfearse1 (ARGT1) responsible for transformation of ATZ residues in rice. Our results showed that transformed lines, when exposed to environmentally realistic ATZ concentration (0.2-0.8 mg/L), displayed significantly high tolerance, with 8-27% biomass and 36-56% chlorophyll content higher, but 37-69% plasma membrane injury lower than untransformed lines. Such results were well confirmed by ARGT1 expression in Arabidopsis. ARGT1-transformed rice took up 1.6-2.7 fold ATZ from its growth medium compared to its wild type (WT) and accumulated ATZ 10%-43% less than that of WT. A long-term study also showed that ATZ in the grains of ARGT1-transformed rice was reduced by 30-40% compared to WT. The ATZ-degraded products were characterized by UPLC/Q-TOF-MS/MS. More ATZ metabolites and conjugates accumulated in ARGT1-transformed rice than in WT. Eight ATZ metabolites for Phase I reaction and 10 conjugates for Phase II reaction in rice were identified, with three ATZ-glycosylated conjugates that have never been reported before. These results indicate that ARGT1 expression can facilitate uptake of ATZ from environment and metabolism in rice plants.

Activity, biomass and composition of microbial communities and their degradation pathways in exposed propazine soil.

Propazine is a s-triazine herbicide widely used for controlling weeds for crop production. Its persistence and contamination in environment nagatively affect crop growth and food safety. Elimination of propazine residues in the environment is critical for safe crop production. This study identified a microbial community able to degrade propazine in a farmland soil. About 94% of the applied propazine was degraded within 11 days of incubation when soil was treated with 10mgkg-1 propazine as the initial concentration. The process was accompanied by increased microbial biomass and activities of soil enzymes. Denaturing gradient gel electrophoresis (DGGE) revealed multiple bacterial strains in the community as well as dynamic change of the composition of microbial community with a reduced microbial diversity (H' from 3.325 to 2.78). Tracking the transcript level of degradative genes AtzB, AtzC and TrzN showed that these genes were induced by propazine and played important roles in the degradation process. The activities of catalase, dehydrogenase and phenol oxidase were stimulated by propazine exposure. Five degradation products (hydroxyl-, methylated-, dimeric-propazine, ammeline and ammelide) were characterized by UPLC-MS2, revealing a biodegradation of propazine in soil. Several novel methylated and dimeric products of propazine were characterized in thepropazine-exposed soil. These data help understand the pathway, detailed mechanism and efficiency of propazine biodegradation in soil under realistic field condition.

Palladium-Catalyzed Cleavage of α-Allenylic Aryl Ether toward Pyrazolemethylene-Substituted Phosphinyl Allenes and Their Transformations via Alkenyl C-P(O) Cleavage.

A palladium-catalyzed two-component coupling of allenylphosphine oxides with conjugated N-tosylhydrazones is revealed. For the first time, the cleavage of α-allenylic aryl ether toward pyrazolemethylene-substituted phosphinyl allenes enabled facile synthesis of combined motifs with pyrazole and allene. Moreover, the obtained adducts could be easily transformed to potential bioactive multifunctionalized phosphinates via a novel alkenyl C-P(O) cleavage.