scholarly journals UPLC-MS/MS method for simultaneously determining nucleosides and methyl-nucleosides in liver mRNA of Epimedin C-induced liver injury mouse model

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Zhizhen Song ◽  
Zeyun Li ◽  
Xueqian Wen ◽  
Ruijuan Liu ◽  
Xin Tian
Keyword(s):  
Liver Injury ◽  
Mouse Model ◽  
High Performance ◽  
Enzymatic Digestion ◽  
Control Group ◽  
Mice Model ◽  
Vacuolar Degeneration ◽  
Serum Aminotransferase ◽  
Mrna Methylation ◽  
Mice Liver

Abstract Background Epimedin C, one of the main active ingredients of Epimedium, has been reported to have potential hepatotoxicity. However, the mechanism of Epimedin C-induced liver injury has not been studied. mRNA methylation, mainly including N6-methyladenosine and N5-methylcytidine, is implicated in the regulation of many biological processes and diseases. The study of quantifying mRNA methylation alterations in Epimedin C-induced liver injury mice may contribute to clarify the mechanism of its hepatotoxicity. Therefore, an analysis method needs to be established to determine nucleoside and methyl-nucleoside levels in liver mRNA. Methods An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to simultaneously determine six nucleosides (adenosine, uridine, cytidine, guanosine, N6-methyladenosine and N5-methylcytidine) in liver mRNA. Besides, the Epimedin C-induced liver injury mouse model was studied by intragastrical administration Epimedin C at a daily dose of 10 or 40 mg/kg for 4 weeks. The nucleoside samples of the mice liver mRNA were prepared and separated on an UPLC column using 0.1% formic acid water and methanol after enzymatic digestion. Then the sample was detected by a Qtrap 6500 mass spectrometer. Results In this method, calibration curves of the six nucleosides showed good linearity over their concentration ranges. The linear ranges were 40–20,000 pg/mL for adenosine, cytidine, N6-methyladenosine and N5-methylcytidine, 0.2–100 ng/mL for guanosine, and 2–1000 ng/mL for uridine. Epimedin C-induced liver injury mouse model was successfully established,which could be proved by the elevation of serum aminotransferase levels, and the increased inflammatory cell infiltration as well as vacuolar degeneration in liver. The N6-methyladenosine and N5-methylcytidine levels, and the ratios of N6-methyladenosine to adenosine and N5-methylcytidine to cytidine of the mice liver mRNA were all significantly increased after Epimedin C treatment. Conclusion The established method was successfully applied to the determination of six nucleosides levels in liver mRNA of the Epimedin C-induced liver injury mice model and the control group. The results indicated that mRNA methylation might be associated with Epimedin C-induced liver injury. This study will facilitate the mechanism research on the hepatotoxicity of Epimedin C.

scholarly journals Development of A LC-MS/MS Method for Simultaneously Determining Nucleosides and Methy-Nucleosides in Mice Liver mRNA of Epimedin C-Induced Liver Injury Model​

2021 ◽  
Author(s):  
Zhizhen Song ◽  
Zeyun Li ◽  
Xueqian Wen ◽  
Ruijuan Liu ◽  
Xin Tian
Keyword(s):  
Liver Injury ◽  
High Performance ◽  
Liver Toxicity ◽  
Enzymatic Digestion ◽  
Control Group ◽  
Good Precision ◽  
Vacuolar Degeneration ◽  
Serum Aminotransferase ◽  
Mrna Methylation ◽  
Mice Liver

Abstract Background Epimedium, the crude drug used in clinical, has been proved to have the potential to cause liver damage in patients. As one of the main active ingredient of Epimedium, Epimedin C is reported to have the potential hepatotoxicity. However, the mechanism of Epimedin C-induced mice liver injury has not been studied. mRNA methylation is implicated in the regulation of many biological processes and diseases. The study of mRNA methylation in Epimedin C-induced liver injury may contribute to clarify the liver toxicity mechanism of Epimedin C. Therefore, an analysis method needs to be established to determine liver mRNA nucleoside and methy-nucleoside levels in epigenetic studies. Methods The Epimedin C groups were intragastrically administered single doses of 10 and 40 mg/kg body weight of Epimedin C for four weeks, and the normal control group were given the same volume of saline. The results of hematoxylin and eosin staining of the liver and serum aminotransferase levels were used to evaluate liver injury. The nucleoside samples of the mice liver mRNA were prepared and separated on an UPLC column using 0.1% formic acid water and methanol after enzymatic digestion. Then the sample was detected by a Qtrap 6500 mass spectrometer. Results In this study, a selective, rapid and sensitive method was established using high performance liquid chromatography-tandem mass spectrometry for the simultaneous determination of six nucleosides (adenosine, uridine, cytidine, guanosine, N6-methyladenosine and N5-methylcytidine) in mRNA. In this method, linear concentrations of adenosine, cytidine, N6-methyladenosine and N5-methylcytidine were 40-20000 pg/mL, guanosine was 0.2–100 ng/mL, and uridine was 2-1000 ng/mL. N6-methyladenosine and N5-methylcytidine modification levels of the mice liver mRNA increased after Epimedin C treatment. Epimedin C led to the elevation of serum aminotransferase levels, and increased the inflammatory cell infiltration and vacuolar degeneration in liver, indicated the liver injury in mice. Conclusion This method was successfully applied to the detection of six liver mRNA nucleosides levels in Epimedin C-induced liver injury with good precision and accuracy. The results indicated that mRNA methylation might be associated with Epimedin C-induced liver injury. This study offers a method for the research on the mechanism of Epimedin C-induced liver injury, and will facilitate the research on the hepatotoxicity of herbal medicine in epigenetic studies.

scholarly journals The Effect of Implant Origin Differences on Peritoneal Endometriosis in an Endometriosis Mouse Model

2018 ◽  
Vol 7 (1) ◽  
pp. 34-40
Author(s):  
Sutrisno Sutrisno ◽  
Sri Andarini ◽  
I Wayan Arsan Wiyasa ◽  
Umi Kulsum ◽  
Noerhamdani Noerhamdani ◽  
...  
Keyword(s):  
Mouse Model ◽  
Ethinyl Estradiol ◽  
Uterine Cavity ◽  
Inflammatory Cells ◽  
Control Group ◽  
Mice Model ◽  
Inflammatory Infiltration ◽  
Presence Data ◽  
Peritoneal Endometriosis ◽  
Group B

Objectives: This study aimed to observe the difference in the area of endometriosis lesions and the histopathology of inflammatory cells and granuloma masses in an endometriosis mouse model treated with endometrial cell implants, endometrioma capsules, and adenomyosis tissue. Materials and Methods: This is an experimental study with posttest-only research design which was conducted with the control group. Thirty-two mice (Mus musculus) were injected with 0.2 mL/mice cyclosporin A and then were divided into three groups which were injected with endometrial tissue from the uterine cavity (group A), endometriosis from endometrioma capsule (group B), and endometriosis from adenomyosis (group C). The injection was done slowly into the peritoneal cavity, 0.1 mL each, and followed by intramuscularly Ethinyl estradiol, 0.2 μG/mice. On the 15th days, mice were dissected to observe the peritoneal endometriosis implant and microscopic examination with hematoxylin-eosin (HE) staining to determine the inflammatory cell infiltration and mass granuloma presence. Data were analyzed using SPSS, version 19. Results: The study obtained that the area of implanted endometriosis lesions in group C covered a larger area of endometriosis implants than other groups (P < 0.05). The peritoneal damage in group C was the most severe based on the Klopfleisch method (P < 0.05), with mass granuloma and massive infiltration of inflammatory cells and fibrous connective tissue formation occurring in muscle tissue. Conclusions: The implantation of adenomyosis cell tissue is the best method to develop mice model of endometriosis based on its inflammatory infiltration, the extent of lesion implant, and granuloma mass

The Protective Effect of Garden Cress Lepidium sativum against Lipopolysaccharide (LPS) Induced Hepatotoxicity in Mice Model

2021 ◽  
Author(s):  
Abdalla A. Sayed ◽  
Ali M. Ali ◽  
Gamal M. Bekhet
Keyword(s):  
Liver Injury ◽  
Biochemical Analysis ◽  
White Male ◽  
Protective Role ◽  
Oxidative Enzymes ◽  
Lepidium Sativum ◽  
Experimental Investigations ◽  
Control Group ◽  
Mice Model ◽  
Histopathological Studies

Background: Lepidium sativum (LS) is a very potent and often used as anti-cancer is largely limited due to the dose-related toxic effects. The present study investigated the protective role of LS that can reduce the liver injury induced by LPS.Methods: Forty white male mice were randomly divided into five groups: the vehicle control group, LPS group, LPS plus LS group, LS pretreated plus LPS group and LS + LPS + LS group. Mice were sacrificed at 2, 4, 8, 16, 24 and 48h. Blood and liver samples were collected for the experimental investigations. Biochemical analysis, histopathological studies and molecular investigation carried out for different groups used. Result: Biochemical analysis for serum AST, ALT, LDL and HDL levels were determined to evaluate liver status. Oxidative stress of liver examined through determination of oxidative enzymes. Furthermore, proinflammatory (IL-6 and TNF-α) and anti-inflammatory (IL-4 and IL-10) cytokines were investigated. Histopathological liver sections were examined to show the alterations due to LPS injection. Biochemical analysis showed a significant modulatory effect of LS on the LPS challenged mice. Histopathological studies showed that LPS caused liver alterations, such as necrosis, infiltrations of neutrophils, sinusoid congestion and hepatocellular degeneration in the liver. These histopathological modulations were significant by LS pretreatment. These findings indicate that LS has a significant hepatoprotective effect on LPS-induced liver injury in mice model.

179 Xenogeneic and Allogeneic Mesenchymal Stem Cell Transplantation for Treatment of Tibial Bone Fracture in Mice

2018 ◽  
Vol 30 (1) ◽  
pp. 229 ◽  
Author(s):  
K. K. Bajwa ◽  
V. Sharma ◽  
S. Saini ◽  
A. Kumar ◽  
A. Thakur ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Bone Healing ◽  
Therapeutic Potential ◽  
Bone Fractures ◽  
Enzymatic Digestion ◽  
Control Group ◽  
Mesenchymal Stem Cell Transplantation ◽  
Tibial Bone ◽  
Alkaline Phosphatase Staining

Among stem cells, mesenchymal stem cells (MSC) are best suited for therapeutic purposes because of their immunomodulatory properties, ability to be isolated from adult animal at any stage, ease of propagation in the laboratory, and so on. The present study was carried out to isolate and characterise MSC from adipose tissue of mouse (Mus musculus), and to test their application for the treatment of fractured tibia bone in mouse. Cattle and buffalo MSC, already cultured and characterised in our laboratory, were used in the present study as xenogeneic MSC to observe the healing in mouse model. Murine Ad-MSC were isolated from mouse inguinal fat pad by enzymatic digestion method and cultured in growth enriching medium in standard culture conditions. To test the therapeutic potential of MSC, 24 mice were divided into 4 groups: control (C), allogeneic (A), cattle xenogeneic (CX), and buffalo xenogeneic (BX) with 6 mice (having tibial bone mechanical fractured) in each group, and had the corresponding MSC cells injected in the fracture area. The control group was not subjected to any kind of MSC treatment. Post-treatment, healing in all groups was examined for 36 days at different intervals (Days 1, 12, 24, and 36) via digital X-ray imaging. A bone healing score was assigned to each mouse per the protocol provided by RUST (Radiographic Union Scale in Tibial bone) fractures. The results of present study showed that murine Ad-MSC were positive for MSC-specific markers CD44, CD90, CD105, and negative for CD34 and CD45 via RT-PCR and immunocytochemistry. The Ad-MSC were also positive for the alkaline phosphatase staining. Statistical analysis, using Proc GLM (SAS Institute Inc., Cary, NC, USA), revealed that the bone healing was significantly different (P < 0.01) between group C (1.708 ± 0.059) and other groups [group A (2.125 ± 0.061), CX (2.167 ± 0.068), BX (2.250 ± 0.068)], suggesting that healing was greater in groups transplanted with MSC compared with control. However, healing between groups transplanted with MSC (A, CX, and BX) was not significantly different (P > 0.05). In conclusion, we have observed the healing potential of MSC in mouse model via allogeneic and xenogeneic MSC transplantation; the healing potential among the A, CX, and BX MSC groups was similar.

scholarly journals Magnesium isoglycyrrhizinate protects against concanavalin A-induced immunological liver injury in a mouse model

2019 ◽  
Vol 27 (3) ◽  
pp. 281-290
Author(s):  
Zhengyan Jiang ◽  
Liang Zheng
Keyword(s):  
Liver Injury ◽  
Mouse Model ◽  
Concanavalin A ◽  
Plasma Levels ◽  
Control Group ◽  
Model Group ◽  
Tnf Α ◽  
Ifn Γ ◽  
Magnesium Isoglycyrrhizinate ◽  
Liver Tissues

Abstract Background: To evaluate the protective effects of magnesium isoglycyrrhizinate on a mouse model of concanavalin A (ConA)-induced immunological liver injury. Materials and Methods: Forty-eight mice were randomly divided into a normal control group, a model group, three dose groups of magnesium isoglycyrrhizinate (12.5, 25, 50 mg/kg) and a dexamethasone group (2.5 mg/kg). Magnesium isoglycyrrhizinate was intraperitoneally injected for 5 consecutive days, and the model of immunological liver injury was established on the fifth day after caudal vein injection of ConA (20 mg/kg). Blood was collected to detect the activities of alanine transaminase (ALT) and aspartate transaminase (AST) as well as the levels of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). The levels of neopterin (NP) and malondialdehyde (MDA) and the activities of myeloperoxidase (MPO) and superoxide dismutase (SOD) in liver tissues were measured, and histopathological changes were observed. Results: The serum levels of ALT and AST in the model group increased. Hepatic lobules had necrotic foci and inflammatory cell infiltration. The plasma levels of TNF-α and IFN-γ increased. In liver tissues, the levels of NP, MDA and MPO rose, but that of SOD decreased. Magnesium isoglycyrrhizinate significantly attenuated the activities of ALT and AST (P<0.05). Histopathological staining showed that inflammation of the liver was relieved significantly. Magnesium isoglycyrrhizinate also decreased the levels of NP, MDA and MPO in liver tissues (P<0.05), raised that of SOD and reduced the plasma levels of TNF-α and IFN-γ (P<0.05). Conclusion: Magnesium isoglycyrrhizinate protected against ConA-induced immunological liver injury in mice, probably through immune regulation and antioxidation.

scholarly journals Deficiency in reverse cholesterol transport in mice augments sepsis

2020 ◽  
Author(s):  
Qian Wang ◽  
Ling Guo ◽  
Dan Hao ◽  
Misa Ito ◽  
Kai-jiang Yu ◽  
...  
Keyword(s):  
Mouse Model ◽  
Reverse Cholesterol Transport ◽  
Scavenger Receptor ◽  
Cecal Ligation ◽  
Protective Role ◽  
Cholesterol Transport ◽  
Mice Model ◽  
Exon 2 ◽  
Normal Metabolism ◽  
Mice Liver

ABSTRACTBackgroundSepsis claims over 215,000 lives and costs $16.7 billion per year in America alone. Recent studies revealed that HDL receptor scavenger receptor BI (SR-BI or Scarb1) plays a critical protective role in sepsis. Using Scarb1I179N mice, a mutant SR-BI mouse model with 90% depletion in hepatic SR-BI, we previously reported that the mutant mice are susceptible to cecal ligation and puncture (CLP)-induced sepsis. However, using a hypo-AlbCreSR-BIfl/fl mouse model, Huby’s group showed that the liver-specific SR-BI KO mice are not more susceptible to CLP-induced sepsis. In this study, we generated a new floxed SR-BI mouse model to clarify the contribution of hepatic SR-BI in sepsis. SR-BI is known as a receptor that plays a key role in reverse cholesterol transport (RCT) by uptaking cholesterol to the liver. So, our established AlbCreSR-BIfl/fl mice (liver-specific SR-BI KO) is an RCT deficiency mice model that can be used to understand the mechanisms of RCT protecting against sepsis and may provide new insight into the pathogenesis of sepsis.Methods and ResultsWe generated SR-BIfl/fl mice by flanking exon 2. We bred the floxed mice with AlbCre mice to generate AlbCreSR-BIfl/fl mice (liver-specific SR-BI KO mice), then the mice were backcrossed to C57BL/6J for 10 generations. As shown in Fig 1, the liver SR-BI expression was normal in SR-BIfl/fl mice as compared to C57BL/6J (B6) mice, but completely depleted in AlbCreSR-BIfl/fl mice. Using this liver-specific SR-BI KO model, we observed that a deficiency in RCT rendered the mice highly susceptible to CLP-induced sepsis as shown by 80% and 14.3% survival of SR-BIfl/fl and AlbCreSR-BIfl/fl mice, respectively. We found aggravated inflammatory cytokine production, altered leukocyte recruitment and slightly increased in the blood and peritoneal bacteria. Moreover, we found RCT deficiency mice increased both free and total cholesterol levels in serum and showed severer hemolysis in AlbCreSR-BIfl/fl mice than SR-BIfl/fl mice during CLP-induced sepsis. Importantly, when we fed AlbCreSR-BIfl/fl mice with probucol to decrease the cholesterol level in serum before performing CLP, the survival rate of AlbCreSR-BIfl/fl mice improved to 88.9%.ConclusionsDeficiency RCT resulting in abnormal metabolism of cholesterol and lipid metabolism is a risk factor in sepsis and maintain normal metabolism of cholesterol may provide a new insight for sepsis therapies.

The lack of effectiveness of hyperbaric oxygenation as a treatment for Leishmania major in a mouse model

2015 ◽  
Vol 60 (2) ◽  
Author(s):  
Ayelet Livneh ◽  
Ilan Youngster ◽  
Yossef El-On ◽  
Matitiahu Berkovitch ◽  
Ibrahim Abu-Kishk
Keyword(s):  
Mouse Model ◽  
Leishmania Major ◽  
Topical Therapy ◽  
Lesion Size ◽  
Control Group ◽  
Mice Model ◽  
Cutaneous Lesions ◽  
Significant Difference ◽  
Group 2 ◽  
Group 1

AbstractWe aimed to study the effectiveness of hyperbaric oxygen therapy (HOT) (100% oxygen at 2 ATA for 70 minutes each session for 20 consecutive days) on BALB/c male mice infected with Leishmania major. Fifty-one mice were assigned to six groups. Group 1 was treated with HOT from 1 day after the inoculation. In Groups 2-5, treatment began when the cutaneous lesions appeared. Group 2 received HOT only, Group 3 received topical therapy with Leshcutan only, Groups 4 and 5 received a combination of HOT and Leshcutan for 5 and 10 days respectively, and Group 6 did not receive any treatment (control group). When comparing the control group with Group 1, treatment with HOT in Group 1 did not significantly affect the time of the appearance of the lesions. In contrast, mice treated with Leshcutan demonstrated a significant difference in lesion size and spleen dimensions as compared to the rest of the mice (p<0.001). The results show that HOT treatment has no positive effect on the course of Leishmaniasis in a BALB/c mice model infected with Leishmania major. Further studies are needed with a mouse model closer to humans and with different HOT protocols.

NorUDCA improves liver injury and glucose sensitivity in a mouse model of obesity and steatosis

2015 ◽  
Vol 53 (05) ◽  
Author(s):  
D Steinacher ◽  
T Claudel ◽  
T Stojakovic ◽  
M Trauner
Keyword(s):  
Liver Injury ◽  
Mouse Model ◽  
Glucose Sensitivity

Effect of HylocereusPolyrhizus Rind Extract Toward Interleukin-1β, Vascular Endothelial Growth Factor Expression, Endometriosis Implant Area

2017 ◽  
Vol 9 (08) ◽  
Author(s):  
YanuarEka P. ◽  
Hendy Hendarto ◽  
Widjiati .
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Treatment Group ◽  
Negative Control Group ◽  
Negative Control ◽  
Control Group ◽  
Vascular Endothelial ◽  
Mice Model ◽  
Endothelial Growth Factor ◽  
Fruit Rind

Retrograde menstruation lead to I Kappa B Kinase (IKK) fosforilation in peritoneum macrophage and cause secretion of proinflammatory cytokine interleukin1β then stimulate endometriosis cell to produce Vascular Endothelial Growth Factor which lead to increasing of endometriosis lession seen as endometriosis implant area. Cytokine secretion was inhibited through prevention of NF-κB activation by dragon red fruit rind extract (Hylocereuspolyrhizus). The aim of this reserach is to know the effect of dragon red fuit rind extract with 0,25; 0,5; and 1 mg/g bodyweight dosage toward IL-1β, VEGF expression and implant area in endometriosis mice model. The design of this experiment was randomized post test only control group design.Endometrios mice model were made in 14 days and split into two group, positive control group and treatment group after two week negative control group and postive control group were given Na-CMC 0,5% solution consequetively, and treatment group were given dragon red fruit extract with different dosage. Signification number for IL-1β is p>0,05, signification number for VEGF is p>0,05, and implant area signification number is p>0,05. Administration of dragon red fruit rind extract can decrease IL-1β, VEGF, and implant area.

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