Lysine Deprivation Suppresses Adipogenesis in 3T3-L1 Cells: A Transcriptome Analysis.

Growing evidence proves that amino acid restriction can reverse obesity by reducing adipose tissue mass. Amino acids are not only the building blocks of proteins but also serve as signaling molecules in multiple biological pathways. The study of adipocytes' response to amino acid level changes is crucial. It has been reported that a low concentration of lysine suppresses lipid accumulation and transcription of several adipogenic genes in 3T3-L1 preadipocytes. However, the detailed lysine-deprivation-induced cellular transcriptomic changes and the altered pathways have yet to be fully studied. Here, using 3T3-L1 cells, we performed RNA sequencing on undifferentiated and differentiated cells, and differentiated cells under a lysine-free environment, and the data were subjected to KEGG enrichment. We found that the differentiation process of 3T3-L1 cells to adipocytes required the large-scale upregulation of metabolic pathways, mainly on the mitochondrial TCA cycle, oxidative phosphorylation, and downregulation of the lysosomal pathway. Single amino acid lysine depletion suppressed differentiation dose dependently. It disrupted the metabolism of cellular amino acids, which could be partially reflected in the changes in amino acid levels in the culture medium. It inhibited the mitochondria respiratory chain and upregulated the lysosomal pathway, which are essential for adipocyte differentiation. We also noticed that cellular interleukin 6 (IL6) expression and medium IL6 level were dramatically increased, which was one of the targets for suppressing adipogenesis induced by lysine depletion. Moreover, we showed that the depletion of some essential amino acids such as methionine and cystine could induce similar phenomena. This suggests that individual amino acid deprivation may share some common pathways. This descriptive study dissects the pathways for adipogenesis and how the cellular transcriptome was altered under lysine depletion.

[Optimization of ethanol reflux extraction process of Ziziphi Spinosae Semen- Schisandrae Sphenantherae Fructus based on network pharmacology combined with response surface methodology].

The present study optimized the ethanol extraction process of Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus drug pair by network pharmacology and Box-Behnken method. Network pharmacology and molecular docking were used to screen out and verify the potential active components of Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus, and the process evaluation indexes were determined in light of the components of the content determination under Ziziphi Spinosae Semen and Schisandrae Sphenantherae Fructus in the Chinese Pharmacopoeia(2020 edition). The analytic hierarchy process(AHP) was used to determine the weight coefficient of each component, and the comprehensive score was calculated as the process evaluation index. The ethanol extraction process of Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus was optimized by the Box-Behnken method. The core components of the Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus drug pair were screened out as spinosin, jujuboside A, jujuboside B, schisandrin, schisandrol, schisandrin A, and schisandrin B. The optimal extraction conditions obtained by using the Box-Behnken method were listed below: extraction time of 90 min, ethanol volume fraction of 85%, and two times of extraction. Through network pharmacology and molecular docking, the process evaluation indexes were determined, and the optimized process was stable, which could provide an experimental basis for the production of preparations containing Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus.

The Effects of Hedgehog Signaling Pathway on the Proliferation and Apoptosis of Melanoma Cells.

BACKGROUND: Studies have found that the abnormality of the Hedgehog signaling pathway is related to the occurrence and development of a variety of tumors, but the effect of this signaling pathway on melanoma cells is still unclear. METHODS: This study aimed to discuss the effect of Hedgehog signaling pathway on the proliferation and apoptosis of human malignant melanoma A375 cells and explore its possible mechanism in the proliferation and apoptosis of melanoma cells. Different concentrations of Hedgehog signaling pathway inhibitor cyclopamine (5, 10, 20 and 40 µM) were used to treat human melanoma A375 cells for 24, 48, and 72 h, and set a blank control group (0 µM). Trypan blue cell counting method was used to detect cell viability. MTT method was used to detect the inhibition rate of cell proliferation. Transwell was used to detect cell invasion, and flow cytometry was used to detect cell apoptosis. RESULTS: Through the trypan blue cell counting method and MTT experiment, it was found that the Hedgehog signaling pathway inhibitor cyclopamine has an inhibitory effect on the proliferation and viability of melanoma A375 cells (P < 0.05), and the proliferation inhibitory effect is enhanced with prolonged action time in a dose- and time-dependent manner. Transwell experiment showed that compared with the blank control group, the invasion and migration ability of the treated melanoma A375 cells are significantly reduced, and the difference is statistically significant (P < 0.05). Cell apoptosis experiment showed that compared with the blank control group, the apoptosis rate of A375 cells is significantly higher after treated by 40 µM cyclopamine for 24 h, and the difference is statistically significant (P < 0.05). Gli1 and Bcl-2 protein are highly expressed in melanoma A375 cells, and their expressions show a downward trend (P < 0.05) after being treated by cyclopamine. CONCLUSION: Cyclopamine inhibits cell proliferation and induces cell apoptosis by downregulating Gli1. Hedgehog signaling pathway can be used as a new target for the treatment of malignant melanoma, and multiple measures can be used to inhibit the signaling pathway to achieve a therapeutic effect.

[Adsorption Characteristics and Mechanism of Cadmium in Water by Alkali and Magnetic Composite Modified Wheat Straw Biochar].

Wheat straw biochar (BC) was modified by KOH and magnetics to generate composited modified biochar (FKC). Based on characterization by scanning electron microscopy-energy dispersive spectrometry (SEM-EDS), Brunauer-Emmett-Teller (BET), Fourier transform infrared (FT-IR), X-ray diffraction (XRD), and magnetic (VSM) techniques, the adsorption characteristics and mechanisms of Cd2+ in water and the effects of temperature, pH value, and dosage on the adsorption characteristics of FKC were studied. The results showed that the modified biochar was loose and porous. The specific surface area of FKC increased by 19.11 times, the number of aromatic and oxygen-containing functional groups such as O-H, C=O, and C=C increased, and a new functional group Fe-O formed compared to the BC. FKC is magnetic, and its magnetization is 8.43 emu·g-1, which can be recycled and reused. The adsorption of Cd2+ by FKC fitted well with the pseudo-second-order kinetic model and the Langmuir model, indicating that chemical adsorption is the main adsorption mechanism. The theoretical maximum equilibrium adsorption capacity of FKC is 23.44 mg·g-1, which is 1.47 times that of BC. The thermodynamic parameters suggested the adsorption of Cd2+ by FKC was a spontaneous and endothermic process. The adsorption capacity increased with an increase of pH in the region 2-8, and a biochar dosage of 10 g·L-1 was used. After three cycles of adsorption-desorption-adsorption, the adsorption capacity of Cd2+ by FKC still reached 17.71 mg·g-1, indicating that FKC has good reusability. These results can provide a theoretical basis for the application of KOH and magnet-modified biochar from wheat straw to remove heavy metals from contaminated wastewater.