Low‑dose ionizing radiation attenuates high glucose‑induced hepatic apoptosis and immune factor release via modulation of a miR‑155‑SOCS1 axis

Abstract

Diabetic liver injury (DLI) can result in several diseases of the liver, including steatohepatitis, liver fibrosis, cirrhosis, and liver cancer. Low‑dose ionizing radiation (LDIR) has hormetic effects in normal/disease conditions. However, whether LDIR has a beneficial effect on DLI has not been assessed previously. MicroRNA (miR)‑155 and its target gene suppressor of cytokine signaling 1 (SOCS1) play critical roles in modulating hepatic proliferation, apoptosis, and immunity. However, whether a miR‑155‑SOCS1 axis is involved in high glucose (HG) induced hepatic damage remains to be determined. In the present study, mouse hepatocyte AML12 cells were treated with 30 mM glucose (HG), 75 mGy X‑ray (LDIR), or HG plus LDIR. The expression levels of miR‑155 and SOCS1 were determined by reverse transcription‑quantitative PCR and western blotting. Additionally, apoptosis was measured using flow cytometry. The release of inflammatory factors, including TNF‑α, IL‑1β, IL‑6, IL‑10, and IFN‑γ, after HG and/or LDIR treatment was detected by ELISA. The results showed that HG may induce hepatic apoptosis by upregulating the levels of miR‑155 and downregulating the levels of SOCS1. HG also stimulated the secretion of TNF‑α, IL‑1β, IL‑6, and IL‑10. However, LDIR blocked the HG‑induced activation of a miR‑155‑SOCS1 axis and suppressed the release of inflammatory factors. These results indicated that a miR‑155‑SOCS1 axis plays a role in HG‑induced liver injury, and LDIR may exert a hepatoprotective effect by regulating the miR‑155‑SOCS1 axis.

Keywords: hepatic injury; high glucose; low‑dose ionizing radiation; microRNA‑155; suppressor of cytokine signaling 1.

Figure 1.

miR-155 expression levels following the treatment with HG and/or LDIR. AML12 cells were treated with HG for 24, 48, 72, 96, or 120 h, with 75 mGy LDIR, or with HG for 48 h followed by LDIR, after which the miR-155 expression levels were determined. (A) HG treatment increased the expression levels of miR-155, which peak at 48 h post-HG treatment. (B) LDIR alone did not affect the expression levels of miR-155. (C) LDIR suppressed the HG-induced miR-155 upregulation. *P<0.05, **P<0.01, ***P<0.001. NS, not significant; HG, high glucose; NG, normal glucose; LDIR, low-dose ionizing radiation; miR, microRNA.

Figure 2.

LDIR attenuated HG or miR-155-induced cell apoptosis. AML12 cells were treated with HG for 48 h, with 75 mGy LDIR, or transfected with miR-155 mimics or miR-155 inhibitor, and cell apoptosis was determined by flow cytometry. (A and B) Relative miR-155 expression levels following transfection with miR-155 mimics and miR-155 inhibitor. (C) LDIR significantly attenuated the HG-induced cell apoptosis. (D) Transfection of miR-155 mimics induced cell apoptosis, which was attenuated by LDIR. (E) Transfection of miR-155 attenuated the HG-induced cell apoptosis. **P<0.01, ***P<0.001. NS, not significant; HG, high glucose; LDIR, low-dose ionizing radiation; miR, microRNA; NC, negative control.

Figure 3.

LDIR attenuates the HG- or miR-155-induced reduction in SOSC1 expression. (A) Bioinformatics analysis showed that the 3′ UTR of SOCS1 contained a likely miR-155 binding site that was conserved in mice. (B) Dual-luciferase assays confirmed that the SOCS1 3′-UTR was a direct target of miR-155 in AML12 cells. (C) Western blotting results indicated that HG suppressed the expression of SOCS1, which was reversed by LDIR. (D) Transfection of miR-155 mimics reduced the expression of SOCS1 and LDIR reversed this. (E) Transfection of miR-155 inhibitor reversed the HG-induced SOCS1 suppression. **P<0.01, ***P<0.001. HG, high glucose; LDIR, low-dose ionizing radiation; miR, microRNA; SOCS1, suppressor of cytokine signaling 1.

Figure 4.

LDIR attenuated the HG-induced release of inflammatory factors through regulation of a miR-155-SOCS1 axis. AML12 cells were treated by HG for 48 h, 75 mGy LDIR, transfected with siSOCS1, or transfected with miR-155 mimics, after which, the release of five inflammatory factors, including TNF-α, IL-1β, IL-6, IL-10, and IFN-γ was detected by ELISA. (A) LDIR suppresses the HG-induced release of TNF-α, IL-1β, IL-6, and IL-10, but did not affect the release of IFN-γ. (B) SOCS1 expression was knocked down by transfection of siSOCS1 in AML12 cells. (C) Knockdown of SOCS1 expression upregulated the release of TNF-α, IL-1β, IL-6, and IL-10. (D) Transfection of miR-155 mimics suppressed the HG-induced release of TNF-α, IL-1β, IL-6, and IL-10. *P<0.05, **P<0.01, ***P<0.001. HG, high glucose; NG, normal glucose; zLDIR, low-dose ionizing radiation; miR, microRNA; SOCS1, suppressor of cytokine signaling 1; si, small interfering; CT, control.

Figure 5.

Proposed model on how LDIR protects against HG-induced liver injury. LDIR, low-dose ionizing radiation; HG, high glucose. HG, high glucose; LDIR, low-dose ionizing radiation; miR, microRNA; SOCS1, suppressor of cytokine signaling 1.