Shuangxinfang Prevents S100A9-Induced Macrophage/Microglial Inflammation to Improve Cardiac Function and Depression-Like Behavior in Rats After Acute Myocardial Infarction

Abstract

Background: Depression is a common complication of cardiovascular disease, which deteriorates cardiac function. Shuangxinfang (psycho-cardiology formula, PCF) was reported to alleviate myocardial ischemia injury and improve depression-like behavior. Interestingly, our previous proteomics study predicted that the protein S100A9 appeared as an important target, and macrophage/microglial inflammation might be involved in the process of PCF improving depression induced by acute myocardial infarction (AMI). This study aims to validate the proteomics results. Methods: AMI rat models were established in vivo, followed by the administration of PCF or ABR-215757 (also named paquinimod, inhibiting S100A9 binding to TLR4) for 5 days. Forced swimming test (FST) and open field test (OFT) were applied to record depression-like behavior, and echocardiography was employed to evaluate cardiac function. Morphological changes of cardiomyocytes were assessed by HE staining and TUNEL staining on day 7 after cardiac surgery, as well as Masson trichrome staining on day 21. Hippocampal neurogenesis was determined by Nissl staining, while 5-hydroxytryptamine (5-HT), tryptophan/kynurenine ratio, and brain-derived neurotrophic factor (BDNF) in the hippocampus were analyzed as biochemical indicators of depression. We employed RT-qPCR, western blotting, and immunofluorescence to detect the expression of pathway-related genes and proteins. Myocardial and hippocampal expression of inflammatory factors were performed by ELISA. The activation of macrophage and microglia was assessed via immunoreaction using CD68 and Iba1, respectively. For in vitro confirmation, BV2 cells were primed with recombinant protein S100A9 and then treated with PCF serum or ferulic acid to determine alterations in microglial inflammation. Results: Rats in the AMI group showed heart function deterioration and depression-like behavior. Coronary ligation not only brought about myocardial inflammation, cell apoptosis, and fibrosis but also reduced the neurogenesis, elevated the tryptophan/kynurenine ratio, and decreased the content of 5-HT. PCF could ameliorate the pathological and phenotypic changes in the heart and brain and inhibit the expression of the S100A9 protein, the activation of the microglial cell, and the secretion of IL-1β and TNF-α raised by AMI. ABR-215757 showed therapeutic effect and molecular biological mechanisms similar to PCF. Treatment with PCF serum or ferulic acid in vitro was proved to efficiently block the hyperactivation of BV2 cells and increment of cytokine contents induced by recombinant protein S100A9. Conclusion: We identify S100A9 as a novel and potent regulator of inflammation in both the heart and brain. Macrophage/microglia inflammation mediated by S100A9 is considered a pivotal pathogenic in depression after AMI and a major pathway for the treatment of PCF, suggesting that PCF is a promising therapeutic candidate for psycho-cardiology disease.

Keywords: S100A9; Shuangxinfang; acute myocardial infarction; epressive disorder; inflammation; macrophages; microglia; traditional Chinese medicine.

FIGURE 1

Cardiac function parameters of rats after myocardial infarction in echocardiography from each group (n = 7). Values were expressed as mean ± SD. LVEF: left ventricular ejection fraction. LVFS: left ventricular fractional shortening. LViDd: left ventricular end-diastolic inner diameter; LViDs: left ventricular end-systolic inner diameter. LVEDV: left ventricular end-diastolic volume. LVESV: left ventricular end-systolic volume. *p < 0.05, ***p < 0.001, *****p < 0.00001, compared with the sham group. #p < 0.05, ##p < 0.01, compared with the AMI group. & p < 0.05, compared with the AMI group.

FIGURE 2

Effect of PCF on AMI-induced pathological changes in the myocardial tissue. (A) H&E staining showed different levels of inflammatory infiltration in the peri-infarct border zone. Scale bar, 40 μm. (B) TUNEL staining showed cardiac apoptosis on 7 days after MI surgery. Green staining of the nucleus indicated apoptosis, and blue staining marked DAPI. (C) Masson trichrome staining of heart slides at 21 days after MI. Red, myocardium; blue, scarred fibrosis. (D) Quantitative analysis of TUNEL-positive cells in the border zone of infarction area. Three separate fields were calculated from each group. (E) Fibrosis area as a percentage (three samples from each group). ***p < 0.001, ****p < 0.0001, compared with the sham group. #p < 0.05, ###p < 0.001, compared with the AMI group. && p < 0.01, &&& p < 0.001, compared with the AMI group.

FIGURE 3

PCF significantly ameliorated depression-like behavior in rats after myocardial infarction. (A) The number of verticality in open field test (OFT). (B) Distance in center. (C) Total distance. (D) Immobility time of rats in forced swimming test (FST). n = 9 per group. **p < 0.01, compared with the sham group. ####p < 0.0001, compared with the AMI group. & p < 0.05, && p < 0.01, &&&& p < 0.0001, compared with the AMI group.

FIGURE 4

The effect of PCF on the neurotransmitter disorder and decreased neurogenesis caused by myocardial infarction. (A) HPLC analysis of 5-HT in the hippocampus of rats (n = 3). (B) Ratio of kynurenine to tryptophan. (C) Nissl’s staining in the dentate gyrus region of hippocampus from different groups. (D) Western blotting detected the protein expression levels of BDNF in the hippocampus (n = 3). 5-HT: 5-hydroxytryptamine; Kyn: kynurenine; Try: tryptophan; BDNF: brain-derived neurotrophic factor; DG: dentate gyrus. *p < 0.05, **p < 0.01, compared with the sham group. #p < 0.05, compared with the AMI group. & p < 0.05, compared with the AMI group.

FIGURE 5

The effect of PCF on the S100A9/NF-κB pathway in the hippocampus and myocardium. (A) The gene expression of S100A9 and NF-κB in the hippocampus and myocardium detected by RT-qPCR. Data were presented as mean ± SD, n = 3 per group. (B) Immunofluorescence analysis of S100A9 in hippocampus tissues. S100A9 immunostaining was shown in green and DAPI in blue. (C) Mean optical density of S100A9-positive cells. Three fields were selected from each slide. ***p < 0.001, compared with the sham group. #p < 0.05, compared with the AMI group. &&&& p < 0.0001, compared with the AMI group.

FIGURE 6

Protein expression of TLR4 and NF-κB detected by western blotting. (A) The protein bands of TLR4 and NF-κB in the hippocampus. β-actin was reused as the loading control for protein TLR4 and NF-κB p65. (B) The protein bands of TLR4 and NF-κB in the myocardium. Data were presented as mean ± SD. n = 3 per group. #p < 0.05, compared with the AMI group.

FIGURE 7

Levels of S100A9, IL-1β, and TNF-α in the hippocampus and myocardium were determined by an ELISA assay. Values were expressed as mean ± SD. n = 6 per group. **p < 0.01, ****p < 0.0001 compared with the sham group. #p < 0.05, ##p < 0.01, ###p < 0.001, compared with the AMI group. & p < 0.05, && p < 0.01, &&&& p < 0.0001, compared with the AMI group.

FIGURE 8

Macrophage activation in the myocardium and microglial activation in the DG region of AMI rats by immunofluorescence staining. (A) Representative images of CD68 positive cells in the myocardium. (B) Quantification of the CD68⁺ cell number. (C) Representative images of Iba1 positive cells in the DG region of the hippocampus. (D) Quantification of the Iba1⁺ cell number. n = 3 slices from each group. *p < 0.05, **p < 0.01, compared with the sham group. ##p < 0.01, compared with the AMI group. & p < 0.05, && p < 0.01, compared with the AMI group.

FIGURE 9

PCF serum reduced the proinflammatory effect of recombinant protein S100A9 in BV2 cells via inhibiting TLR4. (A) Microglia cells were treated with protein S100A9 (0.01,0.02, 0.05, 0.1 μmol) for 6 h. CCK-8 assay was used to detect the viability of BV2 cells. (B) Effect of PCF serum with a concentration of 5%, 10%, and 20% on the viability of BV-2 cells. (C) Resting microglia (a, ×100 magnification; c, ×400 magnification) and activated microglia (b, ×100 magnification; d, ×400 magnification). Morphological changes of BV2 microglia after incubation with S100A9 protein for 6 h (b,d). (D) The ratio of the maximum radius to the minimum radius in BV2 microglia. (E) The level of S100A9, IL-1β, and TNF-α in the culture media was measured with ELISA. Each value represented the mean ± SD of three independent experiments. CS: control serum; CS-L: control serum low dose; CS-M: control serum medium dose; CS-H: control serum high dose; PCF: PCF serum; PCF-L: PCF serum low dose; PCF-M: PCF serum medium dose; PCF-H: PCF serum high dose; C34: TLR4 inhibitor; FA: ferulic acid; *p < 0.05, compared with the control group. #p < 0.05, compared with the S100A9 group. & p < 0.05, compared with the S100A9 group. ※ p < 0.05, compared with the S100A9 group.