Deletion of interleukin-6 alleviated interstitial fibrosis in streptozotocin-induced diabetic cardiomyopathy of mice through affecting TGFβ1 and miR-29 pathways

Abstract

Interleukin 6 (IL-6) has been shown to be an important regulator of cardiac interstitial fibrosis. In this study, we explored the role of interleukin-6 in the development of diabetic cardiomyopathy and the underlying mechanisms. Cardiac function of IL-6 knockout mice was significantly improved and interstitial fibrosis was apparently alleviated in comparison with wildtype (WT) diabetic mice induced by streptozotocin (STZ). Treatment with IL-6 significantly promoted the proliferation and collagen production of cultured cardiac fibroblasts (CFs). High glucose treatment increased collagen production, which were mitigated in CFs from IL-6 KO mice. Moreover, IL-6 knockout alleviated the up-regulation of TGFβ1 in diabetic hearts of mice and cultured CFs treated with high glucose or IL-6. Furthermore, the expression of miR-29 reduced upon IL-6 treatment, while increased in IL-6 KO hearts. Overexpression of miR-29 blocked the pro-fibrotic effects of IL-6 on cultured CFs. In summary, deletion of IL-6 is able to mitigate myocardial fibrosis and improve cardiac function of diabetic mice. The mechanism involves the regulation of IL-6 on TGFβ1 and miR-29 pathway. This study indicates the therapeutic potential of IL-6 suppression on diabetic cardiomyopathy disease associated with fibrosis.

Figure 1. Level of IL-6 in the serum and heart of mice with diabetes mellitus (DM).

(A,B) IL-6 mRNA level in the serum and heart by qRT-PCR. (C,D) IL-6 level in the serum and heart by Immunoassay. Data are expressed as mean ± SEM. N = 3. Ctl, control. ^*P < 0.05 vs Ctl.

Figure 2. Effect of IL-6 knockout on cardiac function of diabetic mice by echocardiography.

(A) Eject fraction (EF). (B) Fractional shortening (FS). (C) E/A ratio. (D) Left ventricular mass (LV Mass). WT, wild-type; IL-6 KO, IL-6 knockout; DM, Diabetes mellitus. Data are expressed as mean ± SEM. N = 7 or 8. *P < 0.05 vs normal WT, ^#P < 0.05 vs DM WT.

Figure 3. Effect of IL-6 knockout on interstitial fibrosis of diabetic mice.

(A,B) mRNA levels of collagen I and collagen III. WT, wild-type; IL-6 KO, IL-6 knockout; DM, Diabetes mellitus. Data are expressed as mean ± SEM. n = 5. ^*P < 0.05 vs normal WT, ^#P < 0.05 vs DM WT. (C) Protein expression of collagen I and collagen III by western blot. WT, wild-type; IL-6 KO, IL-6 knockout; Nor, Normal; DM, Diabetes mellitus. (D) Deposition of collagen in cardiac tissue by Masson’s staining. Scale bar = 100 μm.

Figure 4. Effects of interleukin-6 (IL-6) on the proliferation of cultured cardiac fibroblasts (CFs).

(A) Effects of glucose treatment on the production of IL-6 in CFs by immunoassay. (B,C) Effects of IL-6 treatment on the viability and proliferation of CFs by MTT assay and DNA incorporation assay. (D,E) mRNA levels of collagen I and collagen III in CFs treated with IL-6. Ctl, Control. NG, normal glucose; HG, high glucose. Ctl, control. Data are expressed as mean ± SEM. N = 4. *P < 0.05 vs NG or Ctl.

Figure 5. Effects of IL-6 knockout (IL-6 KO) on the proliferation of cultured cardiac fibroblasts (CFs) stimulated with high glucose (HG).

(A) Cell viability evaluated by MTT assay. (B,C) mRNA levels of collagen I and collagen III. NG, Normal glucose. WT, Wild-type. Data are expressed as mean ± SEM. N = 4. *P < 0.05 vs NG WT, ^#P < 0.05 vs HG WT.

Figure 6. Effects of IL-6 on TGFβ1 production.

(A) Application of IL-6 increased the production of TGFβ1 in cultured cardiac fibroblasts (CFs) from wild type (WT) mice. (B) Knockout of IL-6 inhibited the production of TGFβ1 induced by high glucose (HG) in cultured CFs. (C) Knockout of IL-6 inhibited the production of TGFβ1 in the heart of diabetic mice. (D) Protein expression of TGFβ1 by western blot. Ctl, control; NG, Normal glucose; Nor, Normal; DM, Diabetes mellitus; WT, Wild-type. Data are expressed as mean ± SEM. N = 4. *P < 0.05 vs WT or Ctl; ^#P < 0.05 vs WT (HG or DM).

Figure 7. miR-29 on IL-6 induced collagenI and TGFB1 production in cultured CFs.

(A) miR-29 levels in cultured cardiac fibroblasts (CFs) isolated from wild type (WT) and IL-6 knockout (IL-6 KO) mice. (B) miR-29 levels after IL-6 stimulation and exogenous miR-29/AMO-29 treatment. (C–E) mRNA levels of collagen I, collagen III and TGFβ1 in cultured CFs treated with IL-6 and miR-29/AMO-29. Ctl, Control; NC, Negative control. Data are expressed as mean ± SEM. N = 4. *P < 0.05 vs WT or Ctl; ^#P < 0.05 vs IL-6; ^$P < 0.05 vs IL-6 + miR-29.

Figure 8

Effect of high glucose and IL-6 knockout on the expression of miR-29 in cultured CFs (A) and cardiac tissue (B) Data are expressed as mean ± SEM. N = 4. *P < 0.05 vs NG or WT; ^#P < 0.05 vs HG or DM.

Figure 9. A schematic graph on the signaling pathway of the regulation of IL-6 on cardiac fibrosis and DCM.DCM, diabetic cardiomyopathy.

Blue line with hammer head for “inhibition”; red line with arrow head for “stimulation”.