Gypenosides improve diabetic cardiomyopathy by inhibiting ROS-mediated NLRP3 inflammasome activation

Abstract

NLRP3 inflammasome activation plays an important role in diabetic cardiomyopathy (DCM), which may relate to excessive production of reactive oxygen species (ROS). Gypenosides (Gps), the major ingredients of Gynostemma pentaphylla (Thunb.) Makino, have exerted the properties of anti-hyperglycaemia and anti-inflammation, but whether Gps improve myocardial damage and the mechanism remains unclear. Here, we found that high glucose (HG) induced myocardial damage by activating the NLRP3 inflammasome and then promoting IL-1β and IL-18 secretion in H9C2 cells and NRVMs. Meanwhile, HG elevated the production of ROS, which was vital to NLRP3 inflammasome activation. Moreover, the ROS activated the NLRP3 inflammasome mainly by cytochrome c influx into the cytoplasm and binding to NLRP3. Inhibition of ROS and cytochrome c dramatically down-regulated NLRP3 inflammasome activation and improved the cardiomyocyte damage induced by HG, which was also detected in cells treated by Gps. Furthermore, Gps also reduced the levels of the C-reactive proteins (CRPs), IL-1β and IL-18, inhibited NLRP3 inflammasome activation and consequently improved myocardial damage in vivo. These findings provide a mechanism that ROS induced by HG activates the NLRP3 inflammasome by cytochrome c binding to NLRP3 and that Gps may be potential and effective drugs for DCM via the inhibition of ROS-mediated NLRP3 inflammasome activation.

Keywords: NLRP3 inflammasome; ROS; cytochrome c; diabetic cardiomyopathy; gypenosides; high glucose.

Figure 1

High glucose‐induced H9C2 cell injury and apoptosis. A‐B, LDH (A) and MTS (B) assays were detected in H9C2 cells treated with high glucose for serial hours (6, 12, 24, 36 and 48 h). Mannitol was the control. C, DNA damage was detected through TUNEL in H9C2 cells treated with high glucose for 48 h. D‐E, Representative images of FCM of H9C2 cells treated with high glucose for 48 h (D) and the statistical result of apoptosis cells (E). Data are shown as mean ± SD. *P ≤ .05, **P ≤ .01

Figure 2

High glucose‐induced NLRP3 inflammasome activation in H9C2 cells. A‐D, The levels of NLRP3 (A), ASC (B), caspase‐1 (C) and IL‐1β (D) mRNAs in H9C2 cells were detected by RT‐PCR and normalized to GAPDH. E, The protein level of IL‐1β in medium supernatants of H9C2 cells was analysed by ELISA. F, Medium supernatants (Sup) and cell lysates (Lys) of H9C2 cells were analysed by immunoblotting as indicated in the text. G‐L, The protein levels of NLRP3 (G), ASC (H), pro‐caspase‐1 (I), pro‐IL‐1β (J), mIL‐1β (K) and P20 (L) were normalized to GAPDH. Data are shown as mean ± SD. **P ≤ .01

Figure 3

Silencing NLRP3 or inhibiting caspase‐1 suppressed NLRP3 inflammasome activation and improved cardiomyocyte damage. A, The level of NLRP3 protein in H9C2 cells treated with DR5‐targeted siRNA was analysed by immunoblotting as indicated in the text. B, Silencing NLRP3 inhibited caspase‐1 activation and IL‐1β secretion in the medium supernatants (Sup) and cell Lysates (Lys) of H9C2 cells. C, Supernatants of H9C2 cells treated with DR5‐targeted siRNA were analysed by ELISA for IL‐1β. D‐E, LDH (D) and MTS (E) were used to detect cell damage in H9C2 cells treated with siRNA. F, The levels of NLRP3 inflammasome markers were assayed via immunoblotting in H9C2 cells treated with Z‐YVAD‐FMK (YVAD) for 48 h. G, Supernatants of H9C2 cells were also analysed by ELISA for IL‐1β. H‐I, LDH (H) and MTS (I) were used to detect cell damage in H9C2 cells treated with Z‐YVAD‐FMK (YVAD). Data are shown as mean ± SD. *P ≤ .05, **P ≤ .01, ***P ≤ .001

Figure 4

Inhibiting ROS‐suppressed NLRP3 inflammasome activation and improved cardiomyocyte damage. A, The cytoplasm and mitochondrion of H9C2 cells were analysed by immunoblotting for cytochrome c after treating with high glucose for 48 h. B, ROS release was detected by FCM in H9C2 cells treated with high glucose for 48 h. C, ROS release was detected by FCM in H9C2 cells treated with high glucose and/or NAC for 48 h. D, The levels of NLRP3 inflammasome markers were assayed via immunoblotting in H9C2 cells treated with NAC for 48 h. E‐F, LDH (E) and MTS (F) were used to detect cell damage in H9C2 cells treated with NAC. Data are shown as mean ± SD. *P ≤ .05, **P ≤ .01

Figure 5

Cytochrome c mediated the ROS‐induced NLRP3 inflammasome activation. A, The cytoplasm and mitochondrion of H9C2 cells were analysed by immunoblotting for cytochrome c after treating with high glucose and/or NAC for 48 h. B, The cytoplasm and mitochondrion of H9C2 cells were analysed by immunoblotting for cytochrome c after being treated with high glucose and/or cyclosporin A for 48 h. C, The levels of NLRP3 inflammasome markers were assayed via immunoblotting in H9C2 cells treated with high glucose and/or cyclosporin A for 48 h. D‐E, LDH (D) and MTS (E) were used to detect cell damage in H9C2 cells treated with cyclosporin A. F, H9C2 cell lysates were analysed by CoIP for NLRP3 and cytochrome c as indicated in the text. Data are shown as mean ± SD. *P ≤ .05, **P ≤ .01

Figure 6

Gps improved cardiomyocyte damage. A‐B, LDH (A) and MTS (B) assays were detected in H9C2 cells treated with Gps for 48 h. C‐D, Representative images of FCM of H9C2 cells treated with Gps for 48 h (C) and the statistical result of apoptosis cells (D). Data are shown as mean ± SD. *P ≤ .05, **P ≤ .01, ***P ≤ .001

Figure 7

Gps suppressed NLRP3 inflammasome activation by inhibiting ROS release. A, The levels of NLRP3 inflammasome markers were assayed via immunoblotting in the medium supernatants (Sup) and lysates (Lys) of H9C2 cells treated with Gps for 48 h. B‐C, The levels of IL‐1β (B) and IL‐18 (C) proteins in medium supernatants of H9C2 cells were analysed by ELISA after being treated with Gps for 48 h. D‐E, ROS release was detected by FCM in H9C2 cells treated with Gps for 48 h (D) and the statistical result of positive cells (E). F, The cytoplasm and mitochondrion of H9C2 cells were analysed by immunoblotting for cytochrome c after treating with Gps for 48 h. Data are shown as mean ± SD. **P ≤ .01, ***P ≤ .001

Figure 8

Gps inhibited NLRP3 inflammasome activation and DCM development in vivo. A‐B, Blood glucose (A) and body weight (B) were measured every week for 13 wk after HFD administration. C, Myocardial tissue sections were stained with H&E fluid to detect myocardial injury. D, The level of CRP protein in the serum was analysed by ELISA. E, The levels of NLRP3 inflammasome markers were assayed via immunoblotting in myocardial tissue. F‐G, The levels of IL‐1β (F) and IL‐18 (G) proteins in serum were analysed by ELISA. H‐K, The gene levels of NLRP3 (H), ASC (H), caspase‐1 (G) and IL‐1β (K) were assayed via RT‐PCR in myocardial tissue. Data are shown as mean ± SD. *P ≤ .05, **P ≤ .01, ***P ≤ .001