FGL2 knockdown improves heart function through regulation of TLR9 signaling in the experimental autoimmune myocarditis rats

Abstract

Fibrinogen-like protein 2 (FGL2) is an important immune regulator of both innate and adaptive response. It is present on the surface of macrophages and endothelial cells, and can be constitutively secreted by CD4⁺CD8⁺ T cells. Previous studies showed that FGL2 is a potential target for the treatment of experimental autoimmune myocarditis. However, the molecular mechanism of the roles of FGL2 in experimental autoimmune myocarditis is poorly understood. Here, we silenced FGL2 gene by using FGL2-RNAi lentivirus to reveal the heart function in experimental autoimmune myocarditis rats. We found that the cardiac myosin of pigs' hearts induced Lewis rats to come into being as autoimmune myocarditis. TLR9 was upregulated in the heart of experimental autoimmune myocarditis rats. After primary immunization (21 day), the cardiac function of the myocarditis model group improved (P < 0.05). Significantly, the levels of INF-α and NF-κB in the FGL2-RNAi-treated group were lower compared to those in the myocarditis model (EAM) group (P < 0.05). Notably, the inflammation score correspondence with the protein and mRNA levels of TLR9 in myocardial tissues was markedly reduced compared to that in the EAM group (P < 0.05). These results support a role of FGL2 to alleviate inflammatory situation in the myocardium through regulation of the TLR9 signaling pathway in the experimental autoimmune myocarditis rats.

Keywords: Experimental autoimmune myocarditis; Fibrinogen-like protein 2; Toll receptor 9.

Fig. 1

Western blotting analysis for TLR9 and Fgl2 protein expression in rat myocardial tissue. a TLR9 and Fgl2 protein expression stripe in each group. TLR9 protein expression in myocardial tissue of the EAM group was significantly higher than that of the control group on day 21 after initial immunization. TLR9 protein expression was significantly lower in the RNAi group compared with the EAM group, and also significantly higher in the RNAi group compared with the NC group. Fgl2 protein expression in myocardial tissue of the EAM group was significantly higher than that in the NC group on day 21 after initial immunization. Fgl2 protein expression was significantly lower compared with those in EAM and GFP groups. b β-Actin as control; the TLR9/β-actin and Fgl2/β-actin ratio of NC was considered as 100%. *P < 0.05 vs NC, group, #P < 0.05 vs EAM group

Fig. 2

Histopathology analysis and inflammatory scores. a Inflammatory cell infiltration was not seen 21 days after the initial immunization in the NC group. There were a large number of focal inflammatory cell infiltrates in the GFP group 21 days after the initial immunization. There were also a large number of focal inflammatory cell infiltrates, mainly lymphocytes, accompanied by capillary expansion and myocardial cell necrosis in the EAM group 21 days after the initial immunization; the bar is representative of 50 μm. b Myocardial inflammation alleviated compared with the EAM group in the FGL2-RNAi group 21 days after the initial immunization. *P < 0.05 vs NC group, ^#P < 0.05 vs EAM group

Fig. 3

Quantitative RT-PCR was used to detect the level of TLR9 and CTLA-4 mRNA. TLR9 and CTLA-4 mRNA expressions in myocardial tissue of the EAM group were significantly higher than in the control group on day 21 after initial immunization. The expression level was significantly higher in the RNAi group compared with the control group but was lower compared with the EAM group. *P < 0.05 vs NC group, #P < 0.05 vs EAM group

Fig. 4

The percentage of CD4+CD25+ T cells in rat serum 21 days after T cells in rat serum 21 days after the initial immunization. The percentage of CD4⁺CD25⁺ T cells in rat serum was detected by FCM. There was no statistically significant difference among all groups (^#P > 0.05)

Fig. 5

INF-α, NF-κB, and IL-6 levels in rat serum. Serum INF-α level in different groups. There was no change in IL-6 level 21 days after the initial immunization in the EAM group. *P < 0.05 vs NC group, #P < 0.05 vs EAM group