Kaempferol attenuated cisplatin-induced cardiac injury via inhibiting STING/NF-κB-mediated inflammation

Abstract

Cardiovascular complications have been well documented as the downside to conventional cancer chemotherapy. As a notable side effect of cisplatin, cardiotoxicity represents a major obstacle to the successful treatment of cancer. It has been reported that kaempferol (KPF) possesses cardioprotective and anti-inflammatory qualities. However, the effect of KPF on cardiac damage caused by conventional cancer chemotherapy remains unclear. In this study, we clarified the protective effect of KPF on cisplatin-induced heart injury, and conducted in-depth research on the molecular mechanism underlying this effect. The results showed that KPF protected against cardiac dysfunction and injury induced by cisplatin in vivo. In H9c2 cells, KPF dramatically reduced cispaltin-induced apoptosis and inflammatory response by modulating STING/NF-κB pathway. In conclusion, these results showed that KPF had great potential in attenuating cisplatin-induced cardiac injury. Besides, greater emphasis should be placed in the future on natural active compounds containing KPF with anti-inflammatory effects for the treatment of these diseases.

Keywords: Kaempferol; STING; cisplatin; inflammation.

Figure 1

KPF attenuated cisplatin-induced hypertrophy and cardiac dysfunction. (A, B) Eject fraction (A) and Fractional shortening (B) were measured by echocardiography. (C) The level of serum CK-MB was detected by the ELISA Kit. (D) The represent images were observed for myocardial fiber disorder. Heart tissues were subjected to (H&E). (E, F) RT-qPCR was used to detect the mRNA levels of ANP and BNP. (G) The ratio of heart to body weight (HW/BW) is assumed to reflect cardiac hypertrophy. (n=6 in each group; #, vs. Saline group, #P<0.05, ##P<0.01, ###P<0.001; *, vs. CDDP group, *P<0.05, **P<0.01).

Figure 2

KPF reduced cisplatin-induced cardiomyocytes apoptosis in myocardial tissue. A. The representative images of TUNEL staining and quantitative analysis were used to demonstrate cardiomyocyte apoptosis. B. Western blot analysis and quantitative analysis were used to detect the expression of apoptosis-related protein BAX and anti-apoptosis-related protein BCL-2. (n=6 in each group; #, vs. Saline group, ###P<0.001; *, vs. CDDP group, ***P<0.001).

Figure 3

KPF significantly suppressed cardiac inflammation induced by cisplatin in myocardial tissue. (A) The representative images of immunohistochemistry staining of CD68 and quantitative analysis were used to demonstrate macrophage infiltration. (B-E) RT-qPCR was used to detect the mRNA levels of IL-6 (B), HMGB1 (C), TNF-α (D), and MCP-1 (E). (F, G) The protein contents of IL-6 (F) and TNF-α (G) were detected with the corresponding ELISA Kit. (H) Western blot analysis and quantitative analysis were used to detect the phosphorylation and expression of NF-κB. (n=6 in each group; #, vs. Saline group, #P<0.05, ##P<0.01, ### P<0.001; *, vs. CDDP group, *P<0.05, **P<0.01, ***P<0.001).

Figure 4

KPF inhibited cell death induced by cisplatin in H9c2 cells. (A) After incubation with cisplatin (0.1, 0.5, 1, 5, or 10 μM) for 24 h, MTT assay was performed to detected cell viability in H9c2 cells. (B) Following 1 h pretreatment with KPF (1, 5, or 10 μM), H9c2 cells were incubated with cisplatin (10 μM) for 24 h and then cell viability was measured by MTT assay. (C, D) Following 1 h pretreatment with KPF (1, 5, or 10 μM), H9c2 cells were incubated with cisplatin (10 μM) for 24 h and then cell apoptosis was measured by TUNEL staining. The representative images of TUNEL staining (C) and quantitative analysis (D) were used to perform apoptosis in H9c2 cells. (E, F) Following 1 h pretreatment with KPF (1, 5, or 10 μM), H9c2 cells were incubated with cisplatin (10 μM) for 24 h and then total protein were collected. Western blot analysis and quantitative analysis were used to detect the expression of BAX and BCL2. (n=3 independent experiments; #, vs. Ctr or DMSO group, #P<0.05, ###P<0.001; *, vs. CDDP group, *P<0.05, **P<0.01, ***P<0.001).

Figure 5

KPF inhibited the expression of cytokine induced by cisplatin. A. After being pretreated with KPF (1, 5, or 10 μM) for 1 h, H9c2 cells were incubated with cisplatin (10 μM) for 6 h and then total RNA was collected. RT-qPCR was used to detect the mRNA levels of HMGB1, MCP-1, TNF-α and IL-6. B. Following 1 h pretreatment with KPF (1, 5, or 10 μM), H9c2 cells were incubated with cisplatin (10 μM) for 24 h and then culture medium were collected. The contents of IL-6 and TNF-α were detected by the corresponding ELISA Kit. C. Following 1 h pretreatment with KPF (1, 5, or 10 μM), H9c2 cells were incubated with cisplatin (10 μM) for 1 h and then total protein was collected. Western blot analysis and quantitative analysis were used to detect the phosphorylation and expression of NF-κB. (n=3 independent experiments; #, vs. DMSO group, ##P<0.01, ###P<0.001; *, vs. CDDP group, *P<0.05, **P<0.01, ***P<0.001).

Figure 6

STING was involved in cisplatin-induced inflammation. (A) Western blot analysis and quantitative analysis were used to detect the phosphorylation of STING and TBK1 in cardiac tissue. (n=6 in each group; #, vs. Saline group, ###P<0.001; *, vs. CDDP group, **P<0.01, ***P<0.001). (B) H9c2 cells were incubated with cisplatin (10 μM) for 0.5, 1 h, or 2 h, and then total protein was collected. Western blot analysis and quantitative analysis were used to detect the phosphorylation of STING and TBK1. (C) Following 1 h pretreatment with KPF (1, 5, or 10 μM), H9c2 cells were incubated with cisplatin (10 μM) for 1 h and then total protein was collected. Western blot analysis and quantitative analysis were used to detect the phosphorylation of STING and TBK1. (D) Specific siRNA for STING were transfected into H9c2 cells for 24 h and then total RNA were collected. RT-qPCR was used to detect the mRNA level of STING. (E-H) Transfecting specific siRNA of STING into H9c2 for 24 h, H9c2 cells were pretreated with KPF (10 μM) for 1 h and then incubated with cisplatin (10 μM) for 24 h. Western blot analysis and quantitative analysis were used to detect the expression of BAX and BCL-2 (E) and cell viability (F) was measured by MTT assay. The contents of IL-6 (G) and TNF-α (H) in the culture medium were detected by the corresponding ELISA Kit. (n=3 independent experiments; #, vs. Ctrl or DMSO group, ##P<0.01, ###P<0.001; *, vs. CDDP group, *P<0.05, **P<0.01, ***P<0.001).