Sophocarpine Suppresses NF-κB-Mediated Inflammation Both In Vitro and In Vivo and Inhibits Diabetic Cardiomyopathy

Abstract

Diabetic cardiomyopathy (DCM) is a leading cause of mortality in patients with diabetes. DCM is a leading cause of mortality in patients with diabetes. We used both in vitro and in vivo experiments to investigate the hypothesis that sophocarpine (SPC), a natural quinolizidine alkaloid derived from a Chinese herb, could protect against DCM. We used hyperglycemic myocardial cells and a streptozotocin (STZ)-induced type 1 diabetes mellitus mouse model. SPC protected myocardial cells from hyperglycemia-induced injury by improving mitochondrial function, suppressing inflammation, and inhibiting cardiac apoptosis. The SPC treatment significantly inhibited the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling in high-glucose-stimulated inflammatory responses. Moreover, SPC significantly slowed the development and progression of DCM in STZ-induced diabetic mice. These results show that SPC suppresses NF-κB-mediated inflammation both in vitro and in vivo and may be used to treat DCM.

Keywords: NF-κB; cardiomyopathy—diagnostics; diabetes; inflammation; sophocarpine.

Figure 1

SPC protects against HG-induced inflammatory responses in H9c2 cells. (A) The chemical structure of SPC. (B) Western blot analysis showed that HG stimulation for 12 h remarkebly increased the expression of COL-1, MMP-9, TGF-β, MyHC, and Bax, which was then significantly inhibited by SPC in a dose dependent manner. (C–F) The results of qPCR further confirmed the findings of western blot analysis. (G) TUNEL staining showed that the increased apoptosis of H9c2 cells was effectively attenuated by SPC. Figures are magnified as 100×. (H) Flow cytometry assay confirmed the results of TUNEL staining. CTL, control group; SPC, Sophocarpine; HG, high glucose. *P < 0.05 when compared with the results of control group; **P < 0.01 when compared with the results of control group; ^#P < 0.05 when compared with the results of HG group; ^##P < 0.01 when compared with the results of HG group.

Figure 2

SPC attenuated HG-stimulated mitochondrial dysfunction in H9c2 cells. (A and B) Effects of SPC (1 mM) treatment on ROS production using flow cytometry assay. (C) Western blot analysis showed that HG induction for 12 h significantly increased cytochrome c release and caspase-3/9 activation, which was effectively inhibited by SPC. Moreover, HG stimulation upregulated the expression of pro-apoptotic Bax and downregulated the expression of anti-apoptotic Bcl-2, whereas these changes were attenuated by application with expression. (D) Quantification of the western blot analysis. CTL, control group; SPC, Sophocarpine; HG, high glucose, ROS, reactive oxygen species. *P < 0.05 when compared with the results of control group; **P < 0.01 when compared with the results of control group; ^#P < 0.05 when compared with the results of HG group; ^##P < 0.01 when compared with the results of HG group.

Figure 3

SPC suppressed the activation of the NF-κB signaling in HG-stimulated inflammatory responses in H9c2 cells. (A) The results of western blot analysis showed that incubation with HG for 12 h remarkblely induced IκBα degradation, and this alteration was significantly reversed by SPC treatment. HG stimulation markedly increased the nuclear translocation of NF-κB/p65, which was abolished by SPC treatment (1 mM). (B) Quantification of the western blot analysis. *P < 0.05 when compared with the results of control group; **P < 0.01 when compared with the results of control group; ^#P < 0.05 when compared with the results of HG group; ^##P < 0.01 when compared with the results of HG group. (C and D) The WB analysis showed successful knockdown of p65 using siRNAs. (E–G) WB analysis showed p65 knockdown using siRNA partially mimicked the anti-apoptotic and anti-inflammatory effects of SPC treatment. (H and I) The results of flow cytometry apoptosis assay. (J and K) The results of flow cytometry ROS production assay. *P < 0.05 when compared with the results of the HG group; **P < 0.01 when compared with the results of the HG group; ^#P < 0.05 when compared with the results of the HG + siRNA-P65 group; ^##P < 0.01 when compared with the results of the HG+siRNA-P65 group. CTL, control group; SPC, Sophocarpine; HG, high glucose; ROS, reactive oxygen species.

Figure 4

SPC treatment attenuated diabetes-induced cardiac dysfunction and cardiac remodeling. (A–E) The results of echocardiography demonstrated that mice in the DM group exhibited significant cardiac dysfunction compared to mice in the control group, with significantly decreased E/A velocity ratio, LVEF, FS, and increased value of LVEDD and LVEDD. After SPC treatment, all these parameters improved notably when compared to DM group. (F) The increased blood glucose level induced by DM was effectively inhibited after SPC treatment. (G–I) Serum inflammatory factor (TNF-α, IL-1β, IL-6) levels were also effectively inhibited after SPC treatment. (J and K) H&E staining and Masson trichrome staining (longitudinal view on the left and transverse view on the right) showed obvious structural abnormalities and collagen accumulation in the myocardial tissues from DM group, while SPC treatment remarkably reduced the collagen deposition and fibrosis. (L) The results of α-SMA IHC staining showed a significant increase of α-SMA protein expression in the DM group, and the protein expression level of α-SMA was decreased after SPC treatment. (M) The expression of TGF-β and TNF-α was also decreased after the treatment of SPC. E/A velocity ratio: the ratio of early to late mitral valve flow velocity E/A velocity ratio; LVEF, left ventricular ejection fraction; FS, percentage of fractional shortening; LVESD, left ventricular end-systolic diameter; LVEDD, left ventricular end-diastolic diameter; IHC staining, immunohistochemical staining; CTL, control group; SPC, Sophocarpine; DM, diabetes mellitus group. *P < 0.05 when compared with the results of control group; ^#P < 0.05 when compared with the results of DM group.

Figure 5

SPC mitigated diabetes-induced cardiac inflammation and myocardium apoptosis. (A) TUNEL staining showed significantly increased cell apoptosis (TUNEL-positive cells) in diabetic hearts, which was remarkably mitigated by SPC treatment. Figures are magnified as 100×. (B) The results of western blot analysis revealed a marked elevation in the protein expression of TNF-α in diabetic myocardial tissues compared with the control group. After SPC treatment, the protein expression of TNF-α was significantly reduced. (C) Quantification of the western blot analysis. (D) Heart tissue samples from mice in diabetes mellitus group have significantly higher Collagen I and Collagen III protein expression than samples from the control group, which was remarkably inhibited by SPC treatment. (E) A diagram recapitulating the main findings of this study. CTL, control group; SPC, Sophocarpine; DM, diabetes mellitus group. *P < 0.05 when compared with the results of control group; **P < 0.01 when compared with the results of control group; ^#P < 0.05 when compared with the results of DM group.