Comparative and phylogenetic analysis of the complete chloroplast genomes of 10 Artemisia selengensis resources based on high-throughput sequencing.

BACKGROUND: Artemisia selengensis, classified within the genus Artemisia of the Asteraceae family, is a perennial herb recognized for its dual utility in culinary and medicinal domains. There are few studies on the chloroplast genome of A. selengensis, and the phylogeographic classification is vague, which makes phylogenetic analysis and evolutionary studies very difficult. RESULTS: The chloroplast genomes of 10 A. selengensis in this study were highly conserved in terms of gene content, gene order, and gene intron number. The genome lengths ranged from 151,148 to 151,257 bp and were typical of a quadripartite structure with a total GC content of approximately 37.5%. The chloroplast genomes of all species encode 133 genes, including 88 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Due to the contraction and expansion of the inverted repeats (IR), the overlap of ycf1 and ndhF genes occurred at the inverted repeats B (IRB) and short single copy sequence (SSC) boundaries. According to a codon use study, the frequent base in the chloroplast genome of A. selengensis' third codon position was A/T. The number of SSR repeats was 42-44, most of which were single nucleotide A/T repeats. Sequence alignment analysis of the chloroplast genome showed that variable regions were mainly distributed in single copy regions, nucleotide diversity values of 0 to 0.009 were calculated by sliding window analysis, 8 mutation hotspot regions were detected, and coding regions were more conserved than non-coding regions. Analysis of non-synonymous substitution (Ka) and synonymous substitution (Ks) revealed that accD, rps12, petB, and atpF genes were affected by positive selection and no genes were affected by neutral selection. Based on the findings of the phylogenetic analysis, Artemisia selengensis was sister to the genus Artemisia Chrysanthemum and formed a monophyletic group with other Artemisia genera. CONCLUSIONS: In this research, the present study systematically compared the chloroplast genomic features of A. selengensis and provided important information for the study of the chloroplast genome of A. selengensis and the evolutionary relationships among Asteraceae species.

Helicobacter pylori CagA-mediated ether lipid biosynthesis promotes ferroptosis susceptibility in gastric cancer.

Helicobacter pylori, particularly cytotoxin-associated gene A (CagA)-positive strains, plays a key role in the progression of gastric cancer (GC). Ferroptosis, associated with lethal lipid peroxidation, has emerged to play an important role in malignant and infectious diseases, but the role of CagA in ferroptosis in cancer cells has not been determined. Here, we report that CagA confers GC cells sensitivity to ferroptosis both in vitro and in vivo. Mechanistically, CagA promotes the synthesis of polyunsaturated ether phospholipids (PUFA-ePLs), which is mediated by increased expression of alkylglycerone phosphate synthase (AGPS) and 1-acylglycerol-3-phosphate O-acyltransferase 3 (AGPAT3), leading to susceptibility to ferroptosis. This susceptibility is mediated by activation of the MEK/ERK/SRF pathway. SRF is a crucial transcription factor that increases AGPS transcription by binding to the AGPS promoter region. Moreover, the results demonstrated that CagA-positive cells are more sensitive to apatinib than are CagA-negative cells, suggesting that detecting the H. pylori CagA status may aid patient stratification for treatment with apatinib.

A Small-Molecule Cocktails-Based Strategy in Culture of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have a variety of unique properties, such as stem cell multipotency and immune regulation, making them attractive for use in cell therapy. Before infusion therapy, MSCs are required to undergo tissue separation, purification, and expansion in vitro for a certain duration. During the process of in vitro expansion of MSCs, the influence of culture time and environment can lead to cell senescence, increased heterogeneity, and function attenuation, which limits their clinical applications. We used a cocktail of three small-molecule compounds, ACY (A-83-01, CHIR99021, and Y-27632), to increase the proliferation activity of MSCs in vitro and reduce cell senescence. ACY inhibited the increase in heterogeneity of MSCs and conserved their differentiation potential. Additionally, ACY maintained the phenotype of MSCs and upregulated the expression of immunomodulatory factors. These results suggest that ACY can effectively improve the quantity and quality of MSCs.

ACSL4 contributes to ferroptosis-mediated rhabdomyolysis in exertional heat stroke.

BACKGROUND: Rhabdomyolysis (RM) is a common complication of exertional heat stroke (EHS) and constitutes a direct cause of death. However, the mechanism underlying RM following EHS remains unclear. METHODS: The murine EHS model was prepared by our previous protocol. RNA sequencing is applied to identify the pathological pathways that contribute to RM following EHS. Inhibition of the acyl-CoA synthetase long-chain family member 4 (ACSL4) was achieved by RNA silencing in vitro prior to ionomycin plus heat stress exposure or pharmacological inhibitors in vivo prior to heat and exertion exposure. The histological changes, the iron accumulation, oxidized phosphatidylethanolamines species, as well as histological evaluation and levels of lipid metabolites in skeletal muscle tissues were measured. RESULTS: We demonstrated that ferroptosis contributes to RM development following EHS. Ferroptosis inhibitor ferrostatin-1 administration once EHS onset significantly ameliorated the survival rate of EHS mice from 35.357% to 52.288% within 24 h after EHS (P = 0.0028 compared with control) and markedly inhibited RM development induced by EHS. By comparing gene expression of between sham heat rest (SHR) (n = 3) and EHS (n = 3) mice in the gastrocnemius (Gas) muscle tissue, we identified that Acsl4 mRNA expression is elevated in Gas muscle tissue of EHS mice (P = 0.0038 compared with SHR), so as to its protein levels (P = 0.0001 compared with SHR). Followed by increase in creatine kinase (CK) and myoglobin (MB) levels, the labile iron accumulation, decrease in glutathione peroxidase 4 (GPX4) expression, and elevation of lipid peroxidation products. From in vivo and in vitro experiments, inhibition of Acsl4 significantly improves muscle cell death caused by EHS, thereby ameliorating RM development, followed by reduction in CK and MB levels by 30-40% (P < 0.0001; n = 8-10) and 40% (P < 0.0001; n = 8-10), restoration of GPX4 expression, and decrease in lipid peroxidation products. Mechanistically, ACSL4-mediated RM seems to be Yes-associated protein (YAP) dependent via TEA domain transcription factor1/TEA domain transcription factor4. CONCLUSIONS: These findings demonstrate an important role of ACSL4 in mediating ferroptosis activation in the development of RM following EHS and suggest that targeting ACSL4 may represent a novel therapeutic strategy to limit the skeletal muscle cell death and prevent RM after EHS.

Neutrophils Culture in Collagen Gel System.

Neutrophils (Neu) migrate rapidly to damaged tissue and play critical roles in host defense and tissue homeostasis, including the intestinal epithelia injuries and immune responses. Although their important roles in these diseases, they are challenging to study due to their short life span and the inability to cryopreserve or expand them in vitro. Moreover, the standard cell culturing on plastic plates (two-dimensional (2D) cultures) does not represent the actual microenvironment where cells reside in tissues. In this study, we developed a new three-dimensional (3D) culture system for human and mouse peripheral blood Neu, which is made of hydrogel. The Neu showed much better cell integrity and less cell debris in the 3D culture system compared to that in 2D culture system. Moreover, the 3D culture system was more suitable for the observation of neutrophil extracellular traps (NETs) stimulated by the classical stimulation phorbol ester (PMA), and other damage associated molecular patterns (DAMPs) such as Lipopolysaccharide (LPS)/ATP, interleukin-1 ß (IL-1ß) and tumor necrosis factor α (TNFα) than the 2D culture system. Moreover, NETs phenomenon in 3D culture system is similar to that in vivo. In addition, the 3D culture system was evaluated to co-culturing Neu and other parenchymal cells, such as colon mucosal epithelial cell lines. In conclusion, the 3D culture system could maintain better properties of Neu than that in 2D culture system and it may reduce the gap between in vitro an in vivo experimentations.

Apatinib induced ferroptosis by lipid peroxidation in gastric cancer.

BACKGROUND: Apatinib, a competitive inhibitor of VEGFR2, has anti-angiogenesis and anticancer activities through different mechanisms, but it still cannot fully explain the drug efficacy of apatinib. Ferroptosis, associated with lethal lipid peroxidation, has emerged to play an important role in cancer biology, however, the exact role of ferroptosis in apatinib-mediating anticancer treatment are still not clear. METHODS: The effects of (1S, 3R)-RSL3 and apatinib were evaluated in different GC cell lines and in normal human gastric epithelial cells. Further, the effects of apatinib and inhibition of antioxidant defense enzyme glutathione peroxidase (GPX4) on cell viability, cell death, glutathione (GSH) levels, lipid ROS production, cellular malondialdehyde (MDA) levels and protein expression were evaluated in vitro as well as in a mouse tumor xenograft model. The expression level of GPX4 in GC tissues and paracancerous tissues was measured by immunohistochemistry. RESULTS: (1S, 3R)-RSL3 selectively killed GC cells, but not normal cells. Apatinib induced ferroptosis in GC cells by decreasing cellular GSH levels and increasing lipid peroxidation levels. This effect was blocked by co-incubation with ferrostatin-1, liproxstatin-1, GSH, or vitamin E. Further investigation revealed that apatinib down-regulated GPX4 expression via inhibition of the transcription factors Sterol regulatory element-binding protein-1a (SREBP-1a). Besides, we found that multi-drug resistant GC cells were vulnerable to apatinib-induced GPX4 inhibition. CONCLUSIONS: In summary, we show that apatinib could induce the lipid peroxidation through GPX4 mediated by SREBP-1a, then negatively regulate the GC cell, even the multi-drug-resistant GC cell, ferroptosis.

Optimization of a Rhabdomyolysis Model in Mice With Exertional Heat Stroke Mouse Model of EHS-Rhabdomyolysis.

Exertional heat stroke (EHS) is a life-threatening disease characterized by high mortality and incidence of rhabdomyolysis (RM). It would therefore be valuable to establish a stable EHS-induced RM model that accurately reflects the clinical characteristics of EHS patients and provides an objective animal model for further study of the pathogenesis of RM. In the current study, 8∼9-week-old, male, wild-type C57BL/6J mice, at the stage of sexual maturity, were randomly divided into four groups: the EHS group, the classical heat stroke (CHS) group, the sham heat exercise group, and sham heat rest group. The survival rate of mice was determined under relatively high levels of temperature and humidity (37.5°C, 65% relative humidity (RH); 37.5°C, 70% RH; 39.5°C, 65% RH; and 39.5°C, 70% RH) as well as a high core temperature (Tc; 42, 42.5, and 43°C). Results showed that the environmental condition of 39.5°C and 65% RH was most suitable for EHS modeling. The end point of EHS evaluation was exhaustion or an individual's core temperature reaching 43°C. The survival rate of mice in the EHS group within 24 h under these conditions was 37.34%, which is consistent with the high mortality characteristics noted in EHS patients. Severe RM was observed in the EHS group by H&E staining and transmission electron microscopy. Creatine kinase levels in the EHS group mostly exceeded 10,000 U/L, which was approximately 10 times higher than that in the sham heat rest group. Renal tubules of the EHS group exhibited severe necrosis, and calcium overload in the skeletal muscles of this group was also observed using intravital 2-photon microscopy. In conclusion, we made improvements to a stable EHS-induced RM animal model to truly reflect the clinical characteristics of EHS patients. This new model should be helpful in the further study of RM pathogenesis.

Dexmedetomidine Attenuates Acute Lung Injury Induced by Heatstroke and Improve Outcome.

INTRODUCTION: Dexmedetomidine (DEX) has been demonstrated to inhibit inflammatory response and protect against multiorgan injury in various scenarios. The objectives of the present study were to ascertain whether DEX is able to attenuate acute lung injury (ALI) under heatstroke (HS), and to explore the underlying mechanism. METHODS: Male C57BL/6 mice were exposed to ambient temperature of 39.5 ±â€Š0.2°C until core temperature reach 43°C. DEX or 0.9% saline was injected i.p. immediately. At the end of the experiment, bronchoalveolar lavage fluid (BALF) and lung tissue were harvested. RESULTS: HS induce ALI and pulmonary dysfunction, while DEX treatment could significantly inhibit lung injury and improve respiratory dysfunction under HS. The overall effect was beneficial and improved the 72 h cumulative survival rate of mice with HS. Furthermore, HS significantly elevated the levels of cytokines in BALF, as well as increased the activity of toll-like receptor 4 (TLR4)/MyD88/nuclear factor-κB (NFκB) signaling pathway in lung tissue, while DEX treatment could inhibit such effects. Finally, DEX could upregulate the expression of caveolin 1 downregulated by HS, which may contribute to the inhibition of TLR4/MyD88/NFκB signaling pathway. DISCUSSION: In conclusion, the present results indicated that DEX may protect against lung inflammatory response and injury under HS via TLR4/MyD88/NFκB signaling pathway, and caveolin-1 may participate in the effects.