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. 2018 Mar;22(3):1816-1825.
doi: 10.1111/jcmm.13464. Epub 2018 Jan 5.

H3 relaxin inhibits the collagen synthesis via ROS- and P2X7R-mediated NLRP3 inflammasome activation in cardiac fibroblasts under high glucose

Xiaohui Zhang  1 , Yu Fu  1 , Hui Li  2 , Li Shen  1 , Qing Chang  1 , Liya Pan  1 , Siting Hong  1 , Xinhua Yin  1
Affiliations

H3 relaxin inhibits the collagen synthesis via ROS- and P2X7R-mediated NLRP3 inflammasome activation in cardiac fibroblasts under high glucose

Xiaohui Zhang et al. J Cell Mol Med. 2018 Mar.

Erratum in

Abstract

Excessive production of reactive oxygen species (ROS) and P2X7R activation induced by high glucose increases NLRP3 inflammasome activation, which contributes to the pathogenesis of diabetic cardiomyopathy. Although H3 relaxin has been shown to inhibit cardiac fibrosis induced by isoproterenol, the mechanism has not been well studied. Here, we demonstrated that high glucose (HG) induced the collagen synthesis by activation of the NLRP3 inflammasome, leading to caspase-1 activation, interleukin-1β (IL-1β) and IL-18 secretion in neonatal rat cardiac fibroblasts. Moreover, we used a high-glucose model with neonatal rat cardiac fibroblasts and showed that the activation of ROS and P2X7R was augmented and that ROS- and P2X7R-mediated NLRP3 inflammasome activation was critical for the collagen synthesis. Inhibition of ROS and P2X7R decreased NLRP3 inflammasome-mediated collagen synthesis, similar to the effects of H3 relaxin. Furthermore, H3 relaxin reduced the collagen synthesis via ROS- and P2X7R-mediated NLRP3 inflammasome activation in response to HG. These results provide a mechanism by which H3 relaxin alleviates NLRP3 inflammasome-mediated collagen synthesis through the inhibition of ROS and P2X7R under HG conditions and suggest that H3 relaxin represents a potential drug for alleviating cardiac fibrosis in diabetic cardiomyopathy.

Keywords: NLRP3 inflammasome; P2X7R; ROS; cardiac fibrosis; high glucose.

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Figures

Figure 1
Figure 1
H3 relaxin inhibited the collagen synthesis induced by HG. (A) Collagen I, collagen III and a‐SMA protein expressions were analysed by Western blot. (B) The protein levels of collagen I were normalized to β‐actin (collagen I/β‐actin). (C) The protein levels of collagen III were normalized to β‐actin (collagen III/β‐actin). (D) The protein levels of a‐SMA were normalized to β‐actin (a‐SMA/β‐actin). Data are the means ± S.D., and each measurement carried out six times. *P < 0.05 versus control, **P < 0.01 versus control, # P < 0.05 versus HG, ## P < 0.01 versus HG.
Figure 2
Figure 2
H3 relaxin inhibited NLRP3 inflammasome activation in cardiac fibroblasts. (A) The protein expression of NLRP3 inflammasome markers (NLRP3, cleaved caspase‐1, IL‐1β and IL‐18) was analysed by Western blot. (B) The protein levels of NLRP3 were normalized to β‐actin (NLRP3/β‐actin). (C) The protein levels of cleaved caspase‐1 were normalized to β‐actin (cleaved caspase‐1/β‐actin). (D) The protein levels of IL‐1β were normalized to β‐actin (IL‐1β/β‐actin). (E) The protein levels of IL‐18 were normalized to β‐actin (IL‐18/β‐actin). (F) The protein levels of IL‐1β in cell culture media. (G) The protein levels of IL‐18 in cell culture media. Data are the means ± S.D., and each measurement carried out six times. *P < 0.05 versus control, **P < 0.01 versus control, #P < 0.05 versus HG, ## P < 0.01 versus HG.
Figure 3
Figure 3
H3 relaxin inhibited the collagen synthesis via ROS activation. (A) ROS levels in cardiac fibroblasts were analysed by DCFH. (B) Collagen I, collagen III and a‐SMA protein expressions were analysed by Western blot. (C) The protein levels of collagen I were normalized to β‐actin (collagen I/β‐actin). (D) The protein levels of collagen III were normalized to β‐actin (collagen III/β‐actin). (E) The protein levels of a‐SMA were normalized to β‐actin (a‐SMA/β‐actin). (F) P2X7R protein expressions were analysed by Western blot. (G) The protein levels of P2X7R were normalized to β‐actin (P2X7R/β‐actin). Data are the means ± S.D., and each measurement carried out six times. *P < 0.05 versus control, **P < 0.01 versus control, # P < 0.05 versus HG, ## P < 0.01 versus HG, & P < 0.05 versus HG+H2O2, && P < 0.01 versus HG+H2O2.
Figure 4
Figure 4
H3 relaxin inhibited ROS‐mediated NLRP3 inflammasome activation. (A) The protein expression of NLRP3 inflammasome markers (NLRP3, cleaved caspase‐1, IL‐1β and IL‐18) was analysed by Western blot. (B) The protein levels of NLRP3 were normalized to β‐actin (NLRP3/β‐actin). (C) The protein levels of cleaved caspase‐1 were normalized to β‐actin (cleaved caspase‐1/β‐actin). (D) The protein levels of IL‐1β were normalized to β‐actin (IL‐1β/β‐actin). (E) The protein levels of IL‐18 were normalized to β‐actin (IL‐18/β‐actin). (F)The protein levels of IL‐1β in cell culture media. (G) The protein levels of IL‐18 in cell culture media. Data are the means ± S.D., and each measurement carried out six times. *P < 0.05 versus control, **P < 0.01 versus control, #P < 0.05 versus HG, ## P < 0.01 versus HG, & P < 0.05 versus HG+H2O2, && P < 0.01 versus HG+H2O2.
Figure 5
Figure 5
H3 relaxin inhibited the collagen synthesis via P2X7R activation. (A) P2X7R protein expressions were analysed by Western blot. (B) Collagen I, collagen III and a‐SMA protein expressions were analysed by Western blot. (C) The protein levels of collagen III were normalized to β‐actin (collagen III/β‐actin). (D) The protein levels of collagen I were normalized to β‐actin (collagen I/β‐actin). (E) The protein levels of a‐SMA were normalized to β‐actin (a‐SMA/β‐actin).(F) ROS levels in cardiac fibroblasts were analysed by DCFH. Data are the means ± S.D., and each measurement carried out six times. *P < 0.05 versus control, **P < 0.01 versus control, # P < 0.05 versus HG, ## P < 0.01 versus HG, & P < 0.05 versus HG+BzATP, && P < 0.01 versus HG+BzATP.
Figure 6
Figure 6
H3 relaxin inhibited P2X7R‐mediated NLRP3 inflammasome activation (A) The protein expression of NLRP3 inflammasome markers (NLRP3, cleaved caspase‐1, IL‐1β and IL‐18) was analysed by Western blot. (B) The protein levels of NLRP3 were normalized to β‐actin (NLRP3/β‐actin). (C) The protein levels of cleaved caspase‐1 were normalized to β‐actin (cleaved caspase‐1/β‐actin). (D) The protein levels of IL‐1β were normalized to β‐actin (IL‐1β/β‐actin). (E) The protein levels of IL‐18 were normalized to β‐actin (IL‐18/β‐actin). (F) The protein levels of IL‐1β in cell culture media. (G) The protein levels of IL‐18 in cell culture media. Data are the means ± S.D., and each measurement carried out six times. *P < 0.05 versus control, **P < 0.01 versus control, # P < 0.05 versus HG, ## P < 0.01 versus HG, & P < 0.05 versus HG+BzATP, && P < 0.01 versus HG+BzATP.
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