Protective effects of SOCS3 overexpression in high glucose‑induced lung epithelial cell injury through the JAK2/STAT3 pathway

Abstract

Previous studies have suggested that the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway is involved in hyperglycemia‑induced lung injury. The present study aimed to investigate the roles of suppressor of cytokine signaling3 (SOCS3) in the regulation of JAK2/STAT3 activation following high glucose (HG) treatment in A549 human pulmonary epithelial cells. Cell viability was evaluated using Cell Counting Kit-8 and lactate dehydrogenase assays. HG‑induced inflammatory injury in A549 cells was assessed through the evaluation of interleukin‑6 (IL‑6) and tumor necrosis factor‑α (TNF‑α) levels using ELISA. The protein expression levels of SOCS3, JAK2, STAT3, phosphorylated (p)‑JAK2 and p‑STAT3 were determined using western blot analysis. Cellular viability was significantly decreased, whereas IL‑6 and TNF‑α levels were significantly increased, following HG stimulation of A549 cells. In addition, the protein levels of SOCS3, p‑JAK2 and p‑STAT3 were significantly increased in HG‑treated cells. Treatment with the JAK2/STAT3 inhibitor tyrphostin AG490, or SOCS3 overexpression, appeared to prevent the HG‑induced alterations in protein expression. Furthermore, cellular viability was enhanced, whereas the levels of proinflammatory cytokines were suppressed. These finding suggested the involvement of the SOCS3/JAK2/STAT3 signaling pathway in HG‑induced responses in lung cells. Therefore, it may be hypothesized that the inhibition of the JAK2/STAT3 pathway through SOCS3 overexpression may prevent hyperglycemia‑induced lung injury, and may have therapeutic potential for the treatment of patients with diabetic lung injury.

Figure 1.

Successful overexpression of SOCS3 in human A549 pulmonary epithelial cells. Representative photomicrographs demonstrating successful transfection of A549 cells with (A) empty vector (NG+SOCS3⁻) and (B) pcDNA3.1-SCOS3 expression vector (NG+SOCS3⁺). Magnification, ×200. (C) Representative blot demonstrating SOCS3 expression in A549 cells following transfection. β-actin was used as the loading control. (D) Blots were semi-quantified using densitometry. Data are expressed as the mean ± standard error of the mean of 5 independent experiments. *P<0.05 vs. NG group; ^#P<0.05 vs. NG+SOCS3⁻ group. SOCS, suppressor of cytokine signaling; NG, normal glucose.

Figure 2.

Morphological alterations of human A549 pulmonary epithelial cells following HG exposure. Representative photomicrographs of cells cultured in (A) normal glucose conditions, (B) HG conditions, (C) hyperosmotic conditions, (D) HG conditions + tyrphostin AG490, (E) HG conditions + empty vector and (F) HG conditions + pcDNA3.1-SOCS3 expression vector. Photomicrographs were captured using an inverted microscope. Magnification, ×200. HG, high glucose; SOCS3, suppressor of cytokine signaling.

Figure 3.

Viability of human A549 pulmonary epithelial cells following various treatments evaluated using. (A) Cell Counting Kit-8 and (B) LDH cytotoxicity assays. Cells were cultured under NG, HG and OG conditions, with HG + tyrphostin AG490, and with HG + pcDNA3.1-SOCS3 expression vector. Data are expressed as the mean ± standard error of the mean of six independent measurements. *P<0.05 vs. NG group; ^#P<0.05 vs. HG group; ^$P<0.05 vs. HG+SOCS3⁻ group. LDH, lactate dehydrogenase; NG, normal glucose; HG, high glucose; OG, hyperosmotic group; SOCS3, suppressor of cytokine signaling 3.

Figure 4.

Proinflammatory cytokine levels in human A549 pulmonary epithelial cells following various treatments. (A) IL-6 and (B) TNF-α levels were measured using ELISA. Cells were cultured under NG, HG and OG conditions, with HG + tyrphostin AG490, and with HG + pcDNA3.1-SOCS3 expression vector. Data are expressed as the mean ± standard error of the mean of six independent measurements. *P<0.05 vs. NG group; ^#P<0.05 vs. HG group; ^$P<0.05 vs. HG + SOCS3⁻ group. IL, interleukin; TNF, tumor necrosis factor; NG, normal glucose; HG, high glucose; OG, hyperosmotic group; SOCS3, suppressor of cytokine signaling 3.

Figure 5.

Protein expression levels of p-JAK2, p-STAT3 and SOCS3 in human A549 pulmonary epithelial following various treatments. Protein expression levels of (A) p-JAK2 and total JAK2, (B) p-STAT3 and total STAT3 and (C) SOCS3, were detected using western blot analysis. β-actin was used as the loading control. Blots were semi-quantified using densitometry. Densitometric analysis of (D) p-JAK2, (E) p-STAT3 and (F) SOCS3. Cells were cultured under NG, HG and OG conditions, with HG + tyrphostin AG490, and with HG + pcDNA3.1-SOCS3 expression vector. Data areexpressed as the mean ± standard error of the mean of 10 independent experiments. *P<0.05 vs. NG group; ^#P<0.05 vs. HG group; ^$P<0.05 vs.HG + SOCS3⁻ group. JAK, Janus kinase; NG, normal glucose; HG, high glucose; OG, hyperosmotic group; SOCS3, suppressor of cytokine signaling 3; p-, phosphorylated; STAT, signal transducers and activators of transcription.