Expression of p53 in the Effects of Artesunate on Induction of Apoptosis and Inhibition of Proliferation in Rat Primary Hepatic Stellate Cells

BACKGROUND: Activation of hepatic stellate cells (HSCs) plays an important role in the development of cirrhosis through the increased production of collagen. p53, the "guardian of the genome", is a transcription factor that can bind to promoter regions of hundreds of genes where it either activates or suppresses gene expression. Thereby, p53 serves as a tumor suppressor by inducing cell cycle arrest, apoptosis, senescence and DNA repair. Artesunate is a derivative of Artemisinin, Scholars had found it had more extensive pharmacological effects past 10 years. However, little is known about the expression of p53 in the effects of Artesunate on induction of apoptosis and inhibition of proliferation in rat HSCs. METHODOLOGY/PRINCIPAL FINDINGS: Isolated and cultured rat primary HSCs in the flask for 10 days to make cells activated. HSCs were divided into two groups: experimental groups and control groups, experimental groups included with various concentrations of Artesunate (125, 150, 175, 200, 225 µmol/L) for 24, 48 and 72 hours. Analysis of MTT revealed that activated HSCs treated with various concentrations of Artesunate (150-225 µmol/L) were inhibited on dose and time-effect relationships; Concentration of hydroxyproline in supernatant was detected by digestive method; Analysis of flow cytometry demonstrated that Artesunate could arrest cell cycle in G1 and induce apoptosis; The nuclear morphological changes in apoptotic cells were evaluated with DNA staining by Hoechst 33258 dye; The expression of p53 were up-regulated showed by western blotting and RT-PCR. CONCLUSION: Artesunate could inhibit HSCs proliferation in dose-dependent and time-dependent manners in vitro through increase the expression of p53

Optimal coordination strategy for multiple distributed energy systems considering supply, demand, and price uncertainties

Facing a significant increase in connected distributed energy systems, the optimal coordination strategy among multiple distributed energy systems is vitally important to explore. In this paper, an energy management system is introduced and operated by an energy service company, which is responsible for managing the interaction of multiple distributed energy systems. To optimize the day-ahead scheduling of the distributed energy systems, a coordination scheme with a bilevel framework is proposed. The energy interaction between the energy management system and distributed energy systems contains electricity and heat, which is a Stackelberg problem. Two types of internal price schemes, namely, realtime pricing and time-of-use pricing, are discussed. Moreover, the uncertainties of renewable energy resources, energy demand, and energy prices are considered within both upper-and lower-level problems. The problem is formulated as a nonlinear bilevel robust optimization model and transformed into a single-level mixed-integer linear problem. Numerical cases illustrate how the energy management system coordinates with distributed energy systems and show the effectiveness of the coordination strategy such that all participators benefit from the proposed strategy and create a win-win situation. The model and results can serve as references for the business managers of companies that provide energy services for building clusters. (c) 2021 Elsevier Ltd. All rights reserved

Hepatic stellate cells identification.

<p>A. Freshly isolated HSCs were spherical, had strong refraction and suspended in culture medium (×100). B. 10-day primary cultured HSCs were fully spreading, presenting stellate or polygonal forms, granular cells were significantly reduced (×100). C. Cell purity was detected by flow cytometry: freshly isolated HSCs stained by monoclonal anti-desmin and FITC-anti-IgG, and the the positive cells in freshly isolated HSCs were 95.4%. D. Generation 2 HSCs were stained by DAPI: cell nucleus were stained and presented in blue (×600). E. Generation 2 HSCs were stained by GFAP/IgG-TRITC: positive cells were stained and presented in red (×600). F. Generation 2 HSCs were stained by desmin/IgG-FITC: positive cells were stained and presented in green (×600). G. Generation 2 HSCs were stained by GFAP/IgG-TRITC , desmin/IgG-FITC and DAPI: positive cells were stained and the number of HSCs more than 99% (×600).</p

Effects of Artesunate on secretion of hydroxyproline of HSCs (± s).

<p>Compared with control group,</p><p>*<i>P</i><0.01.</p

Effects of different concentrations of Artesunate on HSCs apoptosis.

<p>A–D: Flow cytometric analysis: Apoptotic index increased with the increasing concentration of Artesunate, apoptosis rate in the control group was (40.73±0.81)%, apoptosis rates of treatment group (artesunate concentrations were 150, 175, 200 µmol / L) were (52.63±0.84)%, (63.97±0.50)%, (66.65±0.99)%, the difference was statistically significant (<i>P</i><0.01). However, HSCs have spontaneous apoptosis without the presence of artesunate. E–H: Hoechst 33258 staining showed apoptosis was induced after Artesunate treatment. In Artesunate groups, nuclei appeared typical morphological changes of apoptosis, some appear condensed chromatin state, a high degree of nuclear chromatin condensation, marginalization, and even serious cleavage fragments, resulting in apoptotic bodies, and the nuclei of the normal control group without significant morphological changes, the nucleus shape rules, to issue uniform fluorescent. A, E: control group; B, F: 150 µmol/L Artesunate group; C, G: 175 µmol/L Artesunate group; D, H: 200 µmol/L Artesunate group.</p

Inhibition rate of Artesunate on HSCs under different concentrations and time (%,± s).

<p>Compared with control group,</p><p>*<i>P</i><0.01; compared with 150 µmol / L,</p>#<p><i>P</i><0.01; compared with 175 µmol / L,</p>△<p><i>P</i><0.01; compared with acted for 24 h,</p>☆<p><i>P</i><0.01; compared with acted for 48 h,</p>&<p><i>P</i><0.01. Inhibition of HSCs which cultured with different concentrations of Artesunate for 24, 48, 72 h significantly increased, compared with the control group and the difference was statistically significant (<i>P</i><0.01). In the treatment groups stimulated for 24 h and 48 h, results showed that the inhibitory effect increased with the concentration increasing (<i>P</i><0.01) and suggested that the inhibitory action of Artesunate on HSCs proliferation was in a dose-dependent manner. In group 175 µmol/L, inhibition increased with time increasing (<i>P</i><0.01), at 72 h, in group 150 µmol/L the inhibition rate reached 92.6%, it suggested that inhibitory action of Artesunate on HSCs proliferation was in a time-dependent manner.</p

Effects of Artesunate on p53 contents in HSC (n = 6).

<p>After HSCs acted by Artesunate 150 µmol/L, 175 µmol/L, 200 µmol/L, Compared with control group, the expression of p53 protein increased significantly in Artesunate group, * <i>P</i><0.05.</p

Effects of Artesunate on P53 mRNA expression (n = 6).

<p>After HSCs acted by Artesunate 150 µmol/L, 175 µmol/L, 200 µmol/L, Compared with control group, the expression of P53 mRNA increased significantly in Artesunate group, * <i>P</i><0.05.</p

Effect of different concentrations of Artesunate on HSCs apoptosis (± s, n = 3).

<p>*<i>P</i><0.01.</p