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Forty specific pathogen-free male BALB/C mice of 6–8 weeks old and weighing 18–20 g, were purchased from the Anhui Experimental Animal Center. The mice were housed in the animal room of Anhui Medical University for 30 days. The temperature was maintained at 20–25 °C, the humidity was controlled in the range of (50% ± 5%, the circadian rhythm of light/dark was manually controlled for 12 hours, and the mice were allowed to eat and drink freely. All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUCUNK1). After a week of adaptive feeding, the mice were randomly divided into a control group, 50 μg/kg per day MC-LR group, a 100 μg/kg per day MC-LR group, and a 200 μg/kg per day MC-LR group, with 10 mice in each group. The mice were fed at 12 o’clock every day for 28 days, and feeding was stopped 24 h before euthanizing the mice (Supplementary Figure S1, available in www.besjournal.com).
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Cells were cultured in 10% fetal bovine serum (GIBCO, USA) and 1% penicillin-streptomycin (Beyotime, China) in RPMI 1640 medium (DMEM) (GIBCO, USA) with a 1% insulin-transferrin-selenium (Beyotime, China) additive. The culture dish was placed in an incubator at 37 °C and 5% CO2, and the medium was changed every 24 hours and the cell growth was observed. After 72 h, the cells were digested and passaged, and the protein, RNA, and supernatants of the third-generation cells were collected and extracted for subsequent experiments.
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The serum of the mice was centrifuged and measured according to the manufacturer’s instructions for aspartate aminotransferase (AST), alanine aminotransferase (ALT), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), and total triglyceride (TG) detection kits (Jiancheng Biology, China). Interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), and interleukin-10 (IL-10) (Calvin, China) levels were analyzed by enzyme-linked immunosorbent assay (ELISA).
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Mouse livers were embedded in paraffin and sectioned after exposure to MC-LR. Paraffin sections of 5 μm were stained with hematoxylin and eosin (Solarbio, China) to observe mouse liver inflammation and balloon-like lesions of hepatocytes.
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Cells were seeded at 1 × 106 cells/well in 6-well plates prior to transfection and transfected when cultured to 70%–90% confluency. According to the manufacturer’s instructions, Lipofectamine 3000 (Thermo Fisher Scientific, USA) plus 5 μg of a pEGFP vector (Sangon Biotech Co., Ltd, China) containing SFRP5 (OV-SFRP5) or the empty vector (OV-NC) were used for transfection, incubated for 48 hours at 37 °C in a 5% CO2 incubator.
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After treatment, cell viability was determined using the CCK-8 assay (Beyotime, China). The cells were seeded in a 96-well plate at a density of 2 × 103 cells/well and incubated overnight at 37 °C. Subsequently, 10 μL of CCK-8 solution was added, and the cells were cultured for 2 h at 37 °C. The absorbance was measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).
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Proteins in the mouse liver and cells were extracted with radioimmunoprecipitation assay lysis buffer (Beyotime, China), and total protein concentration was quantified using a bicinchoninic acid protein assay kit (Beyotime, China). A total of 35 μg of protein was transferred to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then further transferred to a polyvinylidene fluoride membrane (Millipore, USA). They were then incubated with anti-glyceraldehyde-3-phosphate dehydrogenase, anti-SFRP5, anti-Wnt5a, anti-JNK, anti-P-JNK, anti-β-catenin, and anti-Wnt3a (SANTA, USA) for 12 h at 4 °C, followed by incubation with horseradish peroxidase-conjugated anti-mouse antibody for 1 h at 37 °C. The protein bands were visualized by enhanced chemiluminescence detection and analyzed using Image-Pro Plus software.
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Total RNA was extracted from the cells and mouse liver tissues using TRIzol reagent (Invitrogen, USA). Total RNA was reverse transcribed into complementary DNA using PrimeScript™ RT reagent Kit (Roche, China). Quantitative polymerase chain reaction was performed using 1 mL of complementary DNA per well, TaqMan Master Mix (Applied Biosystems), and 20 mmol/L each of the sense and antisense primers. The results were evaluated using the method, and the calculated number of copies was normalized to the number of glyceraldehyde-3-phosphate dehydrogenase mRNA copies in the same sample. The primers used in this study are listed in Table 1.
Table 1. RNA primer
Primer Forward primer (5ʹ–3ʹ) Reverse primer (5ʹ–3ʹ) SREBP-1c TGGAGACATCGCAAACAAG GGTAGACAACAGCCGCATC ACC1 AAGGGACAGTAGAAATCAAA CAGCCTCCAGTAGAAGAAG FASN GCCTCCGTGGACCTTATC ACAGACACCTTCCCGTCA SCD1 GGGAATAGTCAAGAGGCT ACGAGGACGACAATACAA CD36 GGCAGGAGTGCTGGATTA GAGGCGGGCATAGTATCA DGAT1 GTGGGTTCCGTGTTTGC CTCGGTAGGTCAGGTTGTCT SFRP5 CACTGCCACAAGTTCCCCC TCTGTTCCATGAGGCCATCAG Wnt5a CAACTGGCAGGACTTTCTCAA CCTTCTCCAATGTACTGCATGTG GAPDH TCGGAGTCAACGGATTTGGT TGAAGGGGTCATTGATGGCA Note. SREBP-1c, sterol regulatory element-binding transcription factor-1c; ACC1, acetyl-CoA carboxylase 1; FASN, fatty acid synthase; SCD1, stearoyl-CoA desaturase-1; CD36, cluster of differentiation 36; DGAT1, diacylglycerol o-acyltransferase 1; SFRP5, secreted frizzled-related protein 5; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. -
In this study, quantitative variables are expressed as the mean ± standard deviation, GraphPad Prism 8.4 (GraphPad, California, USA) software was used for statistical analysis. One-way analysis of variance was used to evaluate differences between groups. A value of P < 0.05 was considered statistically significant.
doi: 10.3967/bes2024.081
Secreted Frizzled-Related Protein 5 Mediates Wnt5a Expression in Microcystin-Leucine-Arginine-Induced Liver Lipid Metabolism Disorder in Mice
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Abstract:
Objective Microcystin-leucine-arginine (MC-LR) exposure induces lipid metabolism disorders in the liver. Secreted frizzled-related protein 5 (SFRP5) is a natural antagonist of winglesstype MMTV integration site family, member 5A (Wnt5a) and an anti-inflammatory adipocytokine. In this study, we aimed to investigate whether MC-LR can induce lipid metabolism disorders in hepatocytes and whether SFRP5, which has anti-inflammatory effects, can alleviate the effects of hepatic lipid metabolism by inhibiting the Wnt5a/Jun N-terminal kinase (JNK) pathway. Methods We exposed mice to MC-LR in vivo to induce liver lipid metabolism disorders. Subsequently, mouse hepatocytes that overexpressed SFRP5 or did not express SFRP5 were exposed to MC-LR, and the effects of SFRP5 overexpression on inflammation and Wnt5a/JNK activation by MC-LR were observed. Results MC-LR exposure induced liver lipid metabolism disorders in mice and significantly decreased SFRP5 mRNA and protein levels in a concentration-dependent manner. SFRP5 overexpression in AML12 cells suppressed MC-LR-induced inflammation. Overexpression of SFRP5 also inhibited Wnt5a and phosphorylation of JNK. Conclusion MC-LR can induce lipid metabolism disorders in mice, and SFRP5 can attenuate lipid metabolism disorders in the mouse liver by inhibiting Wnt5a/JNK signaling. -
Key words:
- Jun N-terminal kinase /
- Secreted frizzled-related protein 5 /
- Wnt5a /
- Hepatic lipid metabolism disorder
&These authors contributed equally to this work.
注释:1) AUTHOR CONTRIBUTIONS: -
Figure 3. The effects of microcystin-leucine-arginine on serum ALT (A), AST (B), HDL-C (C), LDL-C (D), TC (E), and TG (F) levels in mice (n = 7). MC-LR, microcystin-leucine-arginine; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipid cholesterol; TC, total cholesterol; TG, triglyceride; ALT, alanine transaminase; AST, aspartate transaminase; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significance.
Figure 4. Histological observations of the livers of mice treated with different doses of microcystin-leucine-arginine (200× magnification, scale bar = 150 µm). (A) Hematoxylin and eosin staining observed Inflammatory cell infiltration of mice liver microstructure in various groups. The black arrow indicates infiltrating inflammatory cells. (B) Hematoxylin and eosin staining observed the fat vacuoles of mice liver microstructure in various groups. MC-LR, microcystin-leucine-arginine.
Figure 5. Different groups’ expression levels of the inflammatory factors IL-1β (A), IL-6 (B), and TNF-α (C), and anti-inflammatory factors IL-4 (D) and IL-10 (E) by ELISA (n = 7). MC-LR, microcystin-leucine-arginine; IL-6, interleukin-6; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; IL-4, interleukin-4; IL-10, interleukin-10; ELISA, enzyme-linked immunosorbent assay; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001; ns, no significance.
Figure 6. Different groups’ expression levels of the inflammatory factors IL-6 (A), IL-1β (B), and TNF-α (C), and anti-inflammatory factor IL-4 (D) and IL-10 (E) by ELISA (n = 3). MC-LR, microcystin-leucine-arginine; IL-6, interleukin-6; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; IL-4, interleukin-4; IL-10, interleukin-10; ELISA, enzyme-linked immunosorbent assay; *P < 0.05; **P < 0.01; ****P < 0.001; ns, no significance.
Figure 7. The effects of microcystin-leucine-arginine on the lipid β-oxidation related genes mRNA expression levels in the liver of mice. (A) ACC1 mRNA expression levels in various groups; (B) CD36 mRNA expression levels in various groups; (C) DGAT1 mRNA expression levels in various groups; (D) FASN mRNA expression levels in various groups; (E) SCD1 mRNA expression levels in various groups; (F) SREBP-1c mRNA expression levels in various groups. (n = 7). MC-LR, microcystin-leucine-arginine; SREBP-1c, sterol regulatory element-binding transcription factor-1c; ACC1, acetyl coenzyme A carboxylase 1; FASN, fatty acid synthase; SCD1, stearoyl-CoA desaturase-1; CD36, cluster of differentiation 36; DGAT1, diacylglycerol o-acyltransferase 1; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001; ns, no significance.
Figure 8. (A) Expression of SFRP5 and Wnt5a mRNA in mice with MC-LR-induced liver injury. (B) The effects of MC-LR on the protein expression levels of Wnt5a and SFRP5 in the liver of mice across various groups. SFRP5, secreted frizzled-related protein 5; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MC-LR, microcystin-leucine-arginine; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0. 0001; ns, no significance.
Figure 9. Effects of MC-LR on mRNA and protein expression levels in AML12 cells. Expression levels of Wnt5a and SFRP5 proteins across various groups. SFRP5, secreted frizzle-related protein 5; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MC-LR, microcystin-leucine-arginine; *P < 0.05; ***P < 0. 001; ns, no significance.
Figure 12. Overexpression of SFRP5 inhibits MC-LR-triggered inflammation in AML12 cells. Concentration of inflammatory factors IL-6, IL-1β, and TNF-α in the culture supernatants of AML12 cells (n = 4). MC-LR, microcystin-leucine-arginine; IL-6, interleukin-6; IL-1β,interleukin-1β; TNF-α, tumor necrosis factor-α; *P < 0.05; **P < 0.01; ***P < 0.001; ###P < 0.001; OV, overexpression; NC, negative control; ns, no significance.
Figure 13. Overexpression of SFRP5 represses Wnt5a expression and JNK phosphorylation Representative blots and quantitative analysis of Wnt5a (A), JNK (B), and P-JNK (C). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JNK, Jun N-terminal kinase; P-JNK, phosphorylation-Jun N-terminal kinase; *P < 0.05; ##P < 0.01; OV, overexpression; NC, negative control; ns, no significance.
Table 1. RNA primer
Primer Forward primer (5ʹ–3ʹ) Reverse primer (5ʹ–3ʹ) SREBP-1c TGGAGACATCGCAAACAAG GGTAGACAACAGCCGCATC ACC1 AAGGGACAGTAGAAATCAAA CAGCCTCCAGTAGAAGAAG FASN GCCTCCGTGGACCTTATC ACAGACACCTTCCCGTCA SCD1 GGGAATAGTCAAGAGGCT ACGAGGACGACAATACAA CD36 GGCAGGAGTGCTGGATTA GAGGCGGGCATAGTATCA DGAT1 GTGGGTTCCGTGTTTGC CTCGGTAGGTCAGGTTGTCT SFRP5 CACTGCCACAAGTTCCCCC TCTGTTCCATGAGGCCATCAG Wnt5a CAACTGGCAGGACTTTCTCAA CCTTCTCCAATGTACTGCATGTG GAPDH TCGGAGTCAACGGATTTGGT TGAAGGGGTCATTGATGGCA Note. SREBP-1c, sterol regulatory element-binding transcription factor-1c; ACC1, acetyl-CoA carboxylase 1; FASN, fatty acid synthase; SCD1, stearoyl-CoA desaturase-1; CD36, cluster of differentiation 36; DGAT1, diacylglycerol o-acyltransferase 1; SFRP5, secreted frizzled-related protein 5; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. -
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23396+Supplementary Materials.pdf