1.Isoliquiritigenin induces HMOX1 and GPX4-mediated ferroptosis in gallbladder cancer cells.
Zeyu WANG ; Weijian LI ; Xue WANG ; Qin ZHU ; Liguo LIU ; Shimei QIU ; Lu ZOU ; Ke LIU ; Guoqiang LI ; Huijie MIAO ; Yang YANG ; Chengkai JIANG ; Yong LIU ; Rong SHAO ; Xu'an WANG ; Yingbin LIU
Chinese Medical Journal 2023;136(18):2210-2220
BACKGROUND:
Gallbladder cancer (GBC) is the most common malignant tumor of biliary tract. Isoliquiritigenin (ISL) is a natural compound with chalcone structure extracted from the roots of licorice and other plants. Relevant studies have shown that ISL has a strong anti-tumor ability in various types of tumors. However, the research of ISL against GBC has not been reported, which needs to be further investigated.
METHODS:
The effects of ISL against GBC cells in vitro and in vivo were characterized by cytotoxicity test, RNA-sequencing, quantitative real-time polymerase chain reaction, reactive oxygen species (ROS) detection, lipid peroxidation detection, ferrous ion detection, glutathione disulphide/glutathione (GSSG/GSH) detection, lentivirus transfection, nude mice tumorigenesis experiment and immunohistochemistry.
RESULTS:
ISL significantly inhibited the proliferation of GBC cells in vitro . The results of transcriptome sequencing and bioinformatics analysis showed that ferroptosis was the main pathway of ISL inhibiting the proliferation of GBC, and HMOX1 and GPX4 were the key molecules of ISL-induced ferroptosis. Knockdown of HMOX1 or overexpression of GPX4 can reduce the sensitivity of GBC cells to ISL-induced ferroptosis and significantly restore the viability of GBC cells. Moreover, ISL significantly reversed the iron content, ROS level, lipid peroxidation level and GSSG/GSH ratio of GBC cells. Finally, ISL significantly inhibited the growth of GBC in vivo and regulated the ferroptosis of GBC by mediating HMOX1 and GPX4 .
CONCLUSION
ISL induced ferroptosis in GBC mainly by activating p62-Keap1-Nrf2-HMOX1 signaling pathway and down-regulating GPX4 in vitro and in vivo . This evidence may provide a new direction for the treatment of GBC.
Animals
;
Mice
;
Carcinoma in Situ
;
Chalcones/pharmacology*
;
Ferroptosis
;
Gallbladder Neoplasms/genetics*
;
Glutathione Disulfide
;
Kelch-Like ECH-Associated Protein 1
;
Mice, Nude
;
NF-E2-Related Factor 2/genetics*
;
Reactive Oxygen Species
;
Humans
2.Effect of mitophagy related genes on the antioxidant properties of Saccharomyces cerevisiae.
Wanqi CHENG ; Qianyao HOU ; Chunfeng LIU ; Chengtuo NIU ; Feiyun ZHENG ; Qi LI ; Jinjing WANG
Chinese Journal of Biotechnology 2023;39(8):3464-3480
Mitophagy is a process whereby cells selectively remove mitochondria through the mechanism of autophagy, which plays an important role in maintaining cellular homeostasis. In order to explore the effect of mitophagy genes on the antioxidant activities of Saccharomyces cerevisiae, mutants with deletion or overexpression of mitophagy genes ATG8, ATG11 and ATG32 were constructed respectively. The results indicated that overexpression of ATG8 and ATG11 genes significantly reduced the intracellular reactive oxygen species (ROS) content upon H2O2 stress for 6 h, which were 61.23% and 46.35% of the initial state, respectively. Notable, overexpression of ATG8 and ATG11 genes significantly increased the mitochondrial membrane potential (MMP) and ATP content, which were helpful to improve the antioxidant activities of the strains. On the other hand, deletion of ATG8, ATG11 and ATG32 caused mitochondrial damage and significantly decreased cell vitality, and caused the imbalance of intracellular ROS. The intracellular ROS content significantly increased to 174.27%, 128.68%, 200.92% of the initial state, respectively, upon H2O2 stress for 6 h. The results showed that ATG8, ATG11 and ATG32 might be potential targets for regulating the antioxidant properties of yeast, providing a new clue for further research.
Mitophagy/genetics*
;
Saccharomyces cerevisiae/genetics*
;
Antioxidants
;
Hydrogen Peroxide/pharmacology*
;
Reactive Oxygen Species
3.Mechanism of Qilongtian Capsules in treatment of acute lung injury based on network pharmacology prediction and experimental validation.
Ying XIE ; Xue-Rong SU ; Tong ZHOU ; Yi-Yao LIANG ; Yang-Qian WU ; Yi WAN ; Tu-Lin LU ; Xiao-Li ZHAO ; Zheng-Yan LI
China Journal of Chinese Materia Medica 2023;48(15):4187-4200
This study aimed to explore the mechanism of Qilongtian Capsules in treating acute lung injury(ALI) based on network pharmacology prediction and in vitro experimental validation. Firstly, UPLC-Q-TOF-MS/MS was used to analyze the main chemical components of Qilongtian Capsules, and related databases were used to obtain its action targets and ALI disease targets. STRING database was used to build a protein-protein interaction(PPI) network. Metascape database was used to conduct enrichment analysis of Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG). AutoDock software was used to perform molecular docking verification on the main active components and key targets. Then, the RAW264.7 cells were stimulated with lipopolysaccharide(LPS) for in vitro experiments. Cell viability was measured by MTT and ROS level was measured by DCFH-DA. NO content was measured by Griess assay, and IL-1β, IL-6, and TNF-α mRNA expression was detected by RT-PCR. The predicted targets were preliminarily verified by investigating the effect of Qilongtian Capsules on downstream cytokines. Eighty-four compounds were identified by UPLC-Q-TOF-MS/MS. Through database retrieval, 44 active components with 589 target genes were screened out. There were 560 ALI disease targets, and 65 intersection targets. PPI network topology analysis revealed 10 core targets related to ALI, including STAT3, JUN, VEGFA, CASP3, and MMP9. KEGG enrichment analysis showed that Qilongtian Capsules mainly exerted an anti-ALI effect by regulating cancer pathway, AGE-RAGE, MAPK, and JAK-STAT signaling pathways. The results of molecular docking showed that the main active components in Qilongtian Capsules, including crenulatin, ginsenoside F_1, ginsenoside Rb_1, ginsenoside Rd, ginsenoside Rg_1, ginsenoside Rg_3, notoginsenoside Fe, notoginsenoside G, notoginsenoside R_1, notoginsenoside R_2, and notoginsenoside R_3, had good binding affinities with the corresponding protein targets STAT3, JUN, VEGFA, CASP3, and MMP9. Cellular experiments showed that Qilongtian Capsules at 0.1, 0.25, and 0.5 mg·mL~(-1) reduced the release of NO, while Qilongtian Capsules at 0.25 and 0.5 mg·mL~(-1) reduced ROS production, down-regulated mRNA expression of IL-1β, IL-6, TNF-α, and inhibited the inflammatory cascade. In summary, Qilongtian Capsules may exert therapeutic effects on ALI through multiple components and targets.
Humans
;
Tumor Necrosis Factor-alpha
;
Ginsenosides
;
Caspase 3
;
Matrix Metalloproteinase 9
;
Interleukin-6
;
Molecular Docking Simulation
;
Network Pharmacology
;
Reactive Oxygen Species
;
Tandem Mass Spectrometry
;
Acute Lung Injury/genetics*
;
Capsules
;
RNA, Messenger
;
Drugs, Chinese Herbal/pharmacology*
4.Protective effect of borneol on the cutaneous toxicity of gilteritinib.
Yourong ZHOU ; Yiming YIN ; Xiangliang HUANG ; Yuhuai HU ; Qiaojun HE
Journal of Zhejiang University. Medical sciences 2023;52(5):544-557
OBJECTIVES:
To investigate the effect of borneol on cutaneous toxicity of gilteritinib and to explore possible compounds that can intervene with the cutaneous toxicity.
METHODS:
C57BL/6J male mice were given gilteritinib by continuous gavage for 28 d and the damage to keratinocytes in the skin tissues was observed with hematoxylin and eosin (HE) staining, TUNEL assay and immunohistochemistry. Human keratinocytes HaCaT were treated with gilteritinib, and cell death and morphological changes were examined by SRB staining and microscopy; apoptosis of HaCaT cells was examined by Western blotting, flow cytometry with propidium iodide/AnnexinⅤ double staining and immunofluorescence; the accumulation of cellular reactive oxygen species (ROS) was examined by flow cytometry with DCFH-DA. Compounds that can effectively intervene the cutaneous toxicity of gilteritinib were screened from a natural compound library using SRB method, and the intervention effect of borneol on gilteritinib cutaneous toxicity was further investigated in HaCaT cells and C57BL/6J male mice.
RESULTS:
In vivo studies showed pathological changes in the skin with apoptosis of keratinocytes in the stratum spinosum and stratum granulosum in the modeling group. Invitro studies showed apoptosis of HaCaT cells, significant up-regulation of cleaved poly (ADP-ribose) polymerase (c-PARP) and gamma-H2A histone family member X (γ-H2AX) levels, and increased accumulation of ROS in gilteritinib-modeled skin keratinocytes compared with controls. Screening of the natural compound library revealed that borneol showed excellent intervention effects on the death of HaCaT cells. In vitro, cell apoptosis was significantly reduced in the borneol+gilteritinib group compared to the gilteritinib control group. The levels of c-PARP, γ-H2AX and ROS in cells were significantly decreased. In vivo, borneol alleviated gilteritinib-induced skin pathological changes and skin cell apoptosis in mice.
CONCLUSIONS
Gilteritinib induces keratinocytes apoptosis by causing intracellular ROS accumulation, resulting in cutaneous toxicity. Borneol can ameliorate the cutaneous toxicity of gilteritinib by reducing the accumulation of ROS and apoptosis of keratinocytes in the skin tissue.
Male
;
Humans
;
Animals
;
Mice
;
Reactive Oxygen Species/metabolism*
;
Poly(ADP-ribose) Polymerase Inhibitors/pharmacology*
;
Mice, Inbred C57BL
;
Apoptosis
;
Poly(ADP-ribose) Polymerases/metabolism*
5.Effect of Baicalin on Pyroptosis of Diffuse Large B-Cell Lymphoma Cell Lines DB and Its Mechanism.
Ming LU ; Chun-Ling HE ; Zhen-Tian WU ; Yao LYU ; Xiao-Hui DUAN ; Bing-Xuan WANG ; Shi-Xiong WANG ; Jian-Hong WANG ; Rong LIANG
Journal of Experimental Hematology 2023;31(6):1706-1713
OBJECTIVE:
To investigate the effect of Baicalin on the proliferation and pyroptosis of diffuse large B-cell lymphoma cell line DB and its mechanism.
METHODS:
DB cells were treated with baicalin at different concentrations (0, 5, 10, 20, 40 μmol/L). Cell proliferation was detected by CCK-8 assay and half maximal inhibitory concentration (IC50) was calculated. The morphology of pyroptosis was observed under an inverted microscope, the integrity of the cell membrane was verified by LDH content release assay, and the expressions of pyroptosis-related mRNA and protein (NLRP3, GSDMD, GSDME, N-GSDMD, N-GSDME) were detected by real-time fluorescence quantitative PCR and Western blot. In order to further clarify the relationship between baicalin-induced pyroptosis and ROS production in DB cells, DB cells were divided into control group, baicalin group, NAC group and NAC combined with baicalin group. DB cells in the NAC group were pretreated with ROS inhibitor N-acetylcysteine (NAC) 2 mmol/L for 2 h. Baicalin was added to the combined treatment group after pretreatment, and the content of reactive oxygen species (ROS) in the cells was detected by DCFH-DA method after 48 hours of culture.
RESULTS:
Baicalin inhibited the proliferation of DB cells in a dose-dependent manner (r=-0.99), and the IC50 was 20.56 μmol/L at 48 h. The morphological changes of pyroptosis in DB cells were observed under inverted microscope. Compared with the control group, the release of LDH in the baicalin group was significantly increased (P<0.01), indicating the loss of cell membrane integrity. Baicalin dose-dependently increased the expression levels of NLRP3, N-GSDMD, and N-GSDME mRNA and protein in the pyroptosis pathway (P<0.05). Compared with the control group, the level of ROS in the baicalin group was significantly increased (P<0.05), and the content of ROS in the NAC group was significantly decreased (P<0.05). Compared with the NAC group, the content of ROS in the NAC + baicalin group was increased. Baicalin significantly attenuated the inhibitory effect of NAC on ROS production (P<0.05). Similarly, Western blot results showed that compared with the control group, the expression levels of pyroptosis-related proteins was increased in the baicalin group (P<0.05). NAC inhibited the expression of NLRP3 and reduced the cleavage of N-GSDMD and N-GSDME (P<0.05). Compared with the NAC group, the NAC + baicalin group had significantly increased expression of pyroptosis-related proteins. These results indicate that baicalin can effectively induce pyroptosis in DB cells and reverse the inhibitory effect of NAC on ROS production.
CONCLUSION
Baicalin can inhibit the proliferation of DLBCL cell line DB, and its mechanism may be through regulating ROS production to affect the pyroptosis pathway.
Humans
;
NLR Family, Pyrin Domain-Containing 3 Protein/metabolism*
;
Reactive Oxygen Species/pharmacology*
;
Pyroptosis
;
Cell Line
;
RNA, Messenger
;
Lymphoma, Large B-Cell, Diffuse
6.Anti-oxidant and Anti-inflammatory Effects of Ethanol Extract from Polygala sibirica L. var megalopha Fr. on Lipopolysaccharide-Stimulated RAW264.7 Cells.
Cheng-Liu YANG ; Shi-Bo WANG ; Wen-Ping HE ; Jin-Juan LIU
Chinese journal of integrative medicine 2023;29(10):905-913
OBJECTIVE:
To investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of Polygala sibirica L. var megalopha Fr. (EEP) on RAW264.7 mouse macrophages.
METHODS:
RAW264.7 cells were pretreated with 0-200 µg/mL EEP or vehicle for 2 h prior to exposure to 1 µg/mL lipopolysaccharide (LPS) for 24 h. Nitric oxide (NO) and prostaglandin (PGE2) production were determined by Griess reagent and enzyme-linked immunosorbent assay (ELISA), respectively. The mRNA levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), interleukin-1beta (IL-1β), and IL-6 were determined using reverse transcription polymerase chain reaction (RT-PCR). Western blot assay was used to determine the protein expressions of iNOS, COX-2, phosphorylation of extracellular regulated protein kinases (ERK1/2), c-Jun N-terminal kinase (JNK), inhibitory subunit of nuclear factor Kappa B alpha (Iκ B-α) and p38. Immunofluorescence was used to observe the nuclear expression of nuclear factor-κ B p65 (NF-κ B p65). Additionally, the anti-oxidant potential of EEP was evaluated by reactive oxygen species (ROS) production and the activities of catalase (CAT) and superoxide dismutase (SOD). The 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl (OH), superoxide anion (O2-) radical and nitrite scavenging activity were also measured.
RESULTS:
The total polyphenol and flavonoid contents of EEP were 23.50±2.16 mg gallic acid equivalent/100 g and 43.78±3.81 mg rutin equivalent/100 g. With EEP treatment (100 and 150 µg/mL), there was a notable decrease in NO and PGE2 production induced by LPS in RAW264.7 cells by downregulation of iNOS and COX-2 mRNA and protein expressions (P<0.01 or P<0.05). Furthermore, with EEP treatment (150 µg/mL), there was a decrease in the mRNA expression levels of TNF-α, IL-1β and IL-6, as well as in the phosphorylation of ERK, JNK and p38 mitogen-activated protein kinase (MAPK, P<0.01 or P<0.05), by blocking the nuclear translocation of NF-κ B p65 in LPS-stimulated cells. In addition, EEP (100 and 150 µg/mL) led to an increase in the anti-oxidant enzymes activity of SOD and CAT, with a concomitant decrease in ROS production (P<0.01 or P<0.05). EEP also indicated the DPPH, OH, O2- radical and nitrite scavenging activity.
CONCLUSION
EEP inhibited inflammatory responses in activated macrophages through blocking MAPK/NF-κ B pathway and protected against oxidative stress.
Animals
;
Mice
;
Antioxidants/pharmacology*
;
Lipopolysaccharides/pharmacology*
;
Polygala
;
Transcription Factor RelA/metabolism*
;
Tumor Necrosis Factor-alpha/metabolism*
;
Ethanol/chemistry*
;
Interleukin-6/metabolism*
;
Anti-Inflammatory Agents/chemistry*
;
Reactive Oxygen Species/metabolism*
;
Cyclooxygenase 2/metabolism*
;
Nitrites/metabolism*
;
NF-kappa B/metabolism*
;
Nitric Oxide/metabolism*
;
Superoxide Dismutase/metabolism*
;
RNA, Messenger
;
Nitric Oxide Synthase Type II/metabolism*
7.Network pharmacology and experimental validation of Maxing Shigan decoction in the treatment of influenza virus-induced ferroptosis.
Jiawang HUANG ; Xinyue MA ; Zexuan LIAO ; Zhuolin LIU ; Kangyu WANG ; Zhiying FENG ; Yi NING ; Fangguo LU ; Ling LI
Chinese Journal of Natural Medicines (English Ed.) 2023;21(10):775-788
Influenza is an acute viral respiratory infection that has caused high morbidity and mortality worldwide. Influenza A virus (IAV) has been found to activate multiple programmed cell death pathways, including ferroptosis. Ferroptosis is a novel form of programmed cell death in which the accumulation of intracellular iron promotes lipid peroxidation, leading to cell death. However, little is known about how influenza viruses induce ferroptosis in the host cells. In this study, based on network pharmacology, we predicted the mechanism of action of Maxing Shigan decoction (MXSGD) in IAV-induced ferroptosis, and found that this process was related to biological processes, cellular components, molecular function and multiple signaling pathways, where the hypoxia inducible factor-1(HIF-1) signaling pathway plays a significant role. Subsequently, we constructed the mouse lung epithelial (MLE-12) cell model by IAV-infected in vitro cell experiments, and revealed that IAV infection induced cellular ferroptosis that was characterized by mitochondrial damage, increased reactive oxygen species (ROS) release, increased total iron and iron ion contents, decreased expression of ferroptosis marker gene recombinant glutathione peroxidase 4 (GPX4), increased expression of acyl-CoA synthetase long chain family member 4 (ACSL4), and enhanced activation of hypoxia inducible factor-1α (HIF-1α), induced nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF) in the HIF-1 signaling pathway. Treatment with MXSGD effectively reduced intracellular viral load, while reducing ROS, total iron and ferrous ion contents, repairing mitochondrial results and inhibiting the expression of cellular ferroptosis and the HIF-1 signaling pathway. Finally, based on animal experiments, it was found that MXSGD effectively alleviated pulmonary congestion, edema and inflammation in IAV-infected mice, and inhibited the expression of ferroptosis-related protein and the HIF-1 signaling pathway in lung tissues.
Animals
;
Mice
;
Ferroptosis
;
Network Pharmacology
;
Reactive Oxygen Species
;
Vascular Endothelial Growth Factor A
;
Influenza A virus
;
Iron
;
Hypoxia
8.Muscone inhibits opening of mPTP to alleviate OGD/R-induced injury of HT22 cells.
Ping HUANG ; Mei-Ling YUAN ; Lei WANG ; Yu-Ang CHEN ; Ning WANG ; Si-Peng WU
China Journal of Chinese Materia Medica 2023;48(22):6154-6163
This study aims to investigate the mechanism of muscone in inhibiting the opening of mitochondrial permeability transition pore(mPTP) to alleviate the oxygen and glucose deprivation/reoxygenation(OGD/R)-induced injury of mouse hippocampal neurons(HT22). An in vitro model of HT22 cells injured by OGD/R was established. CCK-8 assay was employed to examine the viability of HT22 cells, fluorescence microscopy to measure the mitochondrial membrane potential, the content of reactive oxygen species(ROS), and the opening of mPTP in HT22 cells. Enzyme-linked immunosorbent assay was employed to determine the level of ATP and the content of cytochrome C(Cyt C) in mitochondria of HT22 cells. Flow cytometry was employed to determine the Ca~(2+) content and apoptosis of HT22 cells. The expression of Bcl-2(B-cell lymphoma-2) and Bcl-2-associated X protein(Bax) was measured by Western blot. Molecular docking and Western blot were employed to examine the binding between muscone and methyl ethyl ketone(MEK) after pronase hydrolysis of HT22 cell proteins. After the HT22 cells were treated with U0126, an inhibitor of MEK, the expression levels of MEK, p-ERK, and CypD were measured by Western blot. The results showed that compared with the OGD/R model group, muscone significantly increased the viability, mitochondrial ATP activity, and mitochondrial membrane potential, lowered the levels of ROS, Cyt C, and Ca~(2+), and reduced mPTP opening to inhibit the apoptosis of HT22 cells. In addition, muscone up-regulated the expression of MEK, p-ERK, and down-regulated that of CypD. Molecular docking showed strong binding activity between muscone and MEK. In conclusion, muscone inhibits the opening of mPTP to inhibit apoptosis, thus exerting a protective effect on OGD/R-injured HT22 cells, which is associated with the activation of MEK/ERK/CypD signaling pathway.
Mice
;
Animals
;
Reactive Oxygen Species/metabolism*
;
Molecular Docking Simulation
;
Apoptosis
;
Oxygen
;
Adenosine Triphosphate/pharmacology*
;
Mitogen-Activated Protein Kinase Kinases/pharmacology*
;
Glucose/metabolism*
9.Nuclear factor-Y mediates pancreatic β-cell compensation by repressing reactive oxygen species-induced apoptosis under metabolic stress.
Siyuan HE ; Xiaoqian YU ; Daxin CUI ; Yin LIU ; Shanshan YANG ; Hongmei ZHANG ; Wanxin HU ; Zhiguang SU
Chinese Medical Journal 2023;136(8):922-932
BACKGROUND:
Pancreatic β-cells elevate insulin production and secretion through a compensatory mechanism to override insulin resistance under metabolic stress conditions. Deficits in β-cell compensatory capacity result in hyperglycemia and type 2 diabetes (T2D). However, the mechanism in the regulation of β-cell compensative capacity remains elusive. Nuclear factor-Y (NF-Y) is critical for pancreatic islets' homeostasis under physiological conditions, but its role in β-cell compensatory response to insulin resistance in obesity is unclear.
METHODS:
In this study, using obese ( ob/ob ) mice with an absence of NF-Y subunit A (NF-YA) in β-cells ( ob , Nf-ya βKO) as well as rat insulinoma cell line (INS1)-based models, we determined whether NF-Y-mediated apoptosis makes an essential contribution to β-cell compensation upon metabolic stress.
RESULTS:
Obese animals had markedly augmented NF-Y expression in pancreatic islets. Deletion of β-cell Nf-ya in obese mice worsened glucose intolerance and resulted in β-cell dysfunction, which was attributable to augmented β-cell apoptosis and reactive oxygen species (ROS). Furthermore, primary pancreatic islets from Nf-ya βKO mice were sensitive to palmitate-induced β-cell apoptosis due to mitochondrial impairment and the attenuated antioxidant response, which resulted in the aggravation of phosphorylated c-Jun N-terminal kinase (JNK) and cleaved caspase-3. These detrimental effects were completely relieved by ROS scavenger. Ultimately, forced overexpression of NF-Y in INS1 β-cell line could rescue palmitate-induced β-cell apoptosis, dysfunction, and mitochondrial impairment.
CONCLUSION
Pancreatic NF-Y might be an essential regulator of β-cell compensation under metabolic stress.
Rats
;
Mice
;
Animals
;
Reactive Oxygen Species/metabolism*
;
Diabetes Mellitus, Type 2/metabolism*
;
Insulin Resistance
;
Insulin
;
Insulin-Secreting Cells/metabolism*
;
Apoptosis
;
Stress, Physiological
;
Transcription Factors/metabolism*
;
Palmitates/pharmacology*
;
Obesity/metabolism*
10.Synergistic effect of β-thujaplicin and tigecycline against tet(X4)-positive Escherichia coli in vitro.
Muchen ZHANG ; Huangwei SONG ; Zhiyu ZOU ; Siyuan YANG ; Hui LI ; Chongshan DAI ; Dejun LIU ; Bing SHAO ; Congming WU ; Jianzhong SHEN ; Yang WANG
Chinese Journal of Biotechnology 2023;39(4):1621-1632
The widespread of tigecycline resistance gene tet(X4) has a serious impact on the clinical efficacy of tigecycline. The development of effective antibiotic adjuvants to combat the looming tigecycline resistance is needed. The synergistic activity between the natural compound β-thujaplicin and tigecycline in vitro was determined by the checkerboard broth microdilution assay and time-dependent killing curve. The mechanism underlining the synergistic effect between β-thujaplicin and tigecycline against tet(X4)-positive Escherichia coli was investigated by determining cell membrane permeability, bacterial intracellular reactive oxygen species (ROS) content, iron content, and tigecycline content. β-thujaplicin exhibited potentiation effect on tigecycline against tet(X4)-positive E. coli in vitro, and presented no significant hemolysis and cytotoxicity within the range of antibacterial concentrations. Mechanistic studies demonstrated that β-thujaplicin significantly increased the permeability of bacterial cell membranes, chelated bacterial intracellular iron, disrupted the iron homeostasis and significantly increased intracellular ROS level. The synergistic effect of β-thujaplicin and tigecycline was identified to be related to interfere with bacterial iron metabolism and facilitate bacterial cell membrane permeability. Our studies provided theoretical and practical data for the application of combined β-thujaplicin with tigecycline in the treatment of tet(X4)-positive E. coli infection.
Humans
;
Tigecycline/pharmacology*
;
Escherichia coli/metabolism*
;
Reactive Oxygen Species/therapeutic use*
;
Plasmids
;
Anti-Bacterial Agents/metabolism*
;
Escherichia coli Infections/microbiology*
;
Bacteria/genetics*
;
Microbial Sensitivity Tests

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