Related proteins and immunoinflammatory phenotype predicted by previous proteomics were examined in this study. Our research highlighted that PCF inhibited macrophage/microglia inflammation by the suppression of S100A9/TLR4/NF-κB signaling after AMI, thus improving cardiac function and depression-like behavior. PCF serum, as well as ferulic acid, alleviated microglia inflammation in vitro.
According to proteomics results from our previous studies, we speculated that PCF may regulate S100A9 over-activation of macrophage/microglia inflammation, leading to a mitigation in subsequent inflammatory processes involved in AMI. The dramatic cardiomyocyte death initiates a cascade of inflammation in AMI, in the process of which, the role of alarmin S100A9 in deteriorating cardiac function has become a hot topic supported by several top journals of clinical and experimental evidence in these years[37–39]. S100A9, as a potent activator of the innate immune response, as well as the damage-associated molecular pattern (DAMP) protein, is abundantly expressed in neutrophils, and rapidly released from activated neutrophils, monocytes/macrophages and dying cardiomyocytes, into the coronary and systemic circulation after myocardial ischaemia. S100A9 interacted locally with toll-like receptor 4 (TLR4) or receptor of advanced glycation endproducts (RAGE) to promote the expression of NF-κB and release IL-1β and TNF-α[41, 42]. The regulatory role of S100A9 in macrophage activation has been brought into focus. The continuous activation of macrophages might be actuated by S100A9 protein, which acts as a character at the center of the stage to orchestrate the functions of the individual players, in cooperation with other proinflammatory cytokines[39, 43]. Recently, Stankiewicz and colleagues analyzed the hippocampal transcriptome of mice subjected to acute and chronic social stress of different duration, and found that hippocampal S100A9 mRNA increased. In addition, central injection of recombinant S100A9 proteins could evoke depressive-like behaviors, TLR4/NF-κB signaling activation as well as microglia activation, the effects of which were attenuated by TLR4 inhibitor TAK-242, indicating that the dysfunction of S100A9/TLR4 signaling in the hippocampus could generate neuroinflammation and depressive-like behaviors. In vitro studies also showed that S100A9 observably increased the secretion of proinflammatory cytokines including TNF-α and IL-6 in cultured BV-2 microglial cells, the process of which was suppressed by TLR4 inhibitors. Microglia activation is not only a hallmark of neuroinflammation, but also contributes to the development of depressive-like behaviors. Recent studies demonstrated that impairment of the normal structure and function of microglia, caused by either intense inflammatory activation can result in depression and associated impairments in neuroplasticity and neurogenesis. Accordingly, some forms of depression can be recognized as a microglial disease (microgliopathy). Similar to the above results, in AMI-induced depressive rats, the level of S100A9 showed an increasing trend in the myocardium and the hippocampus, accompanied by the activation of transcription factor NF-κB and the release of proinflammatory factors. Also, our research showed a higher content of S100A9 in the myocardium and the hippocampus by ELISA in the AMI group. Coronary ligation promoted the activation of macrophage/microglia, respectively evidenced by an increasement in the number of myocardial CD68 positive cells and hippocampal IBA1 positive cells. Intragastric administration of PCF down-regulated the expression of S100A9 and other inflammatory factors, as well as inhibited the activation of microglia. Our results revealed that PCF intervention inhibited inflammation, which might partly attribute to a reduction in the content of S100A9 and the inhibiting effect of macrophage/microglia activation.
For additional verification of the mechanism, ABR-215757 (paquinimod) was used for the inhibition of S100A9. Paquinimod exerted consistent and robust immunomodulatory effects on systemic lupus erythematosus, which has been positively evaluated in a phase 2 randomized controlled trial. The application range of paquinimod has gradually expanded in preclinical studies, mainly lie in its inhibition of inflammatory reaction by blocking the interaction with TLR4 and RAGE[25, 48]. Paquinimod is second generation quinoline-3-carboxamides which show structural similarity to kynurenines, and might be a novel promising therapeutic way for depressive disorder. At the moment, in vivo studies have demonstrated that ABR-215757 effectively ameliorates depressive symptoms. In our research, after continuous administration of ABR-215757 in the whole acute phase, the expression level of S100A9 was significantly down-regulated, as well as NF-κB, IL-1β and TNF-α. Moreover, inhibition of macrophage/microglia activation by ABR-215757 was shown to alleviate inflammation and to modulate 5-HT metabolism. As a result of TLR4 signaling blocking, the depressive-like behaviors were successfully rescued and cardiac function was partially restored by ABR-215757 treatment.
However, it’s still not clearly identified that S100A9 induced an inflammatory response via the TLR4 receptor, nor that PCF inhibits microglial inflammation through this pathway. Therefore, we conducted cell experiments, in which BV2 microglia was stimulated by recombinant S100A9 protein at a concentration of 0.1mmol to construct model groups. Our results showed that S100A9 could induce the release of IL-1β and TNF-α in microglial cells. Cellular morphology revealed the characters of recombinant S100A9 in the activation of microglia. An increase in IL-1β and TNF-α levels derived from activated microglia may promote the depressive symptoms. In addition, the C34 (TLR4 inhibitor) group and PCF (PCF serum) group were set up to elucidate the mechanisms that in vivo studies have failed to elucidate. The inhibition of TLR4 attenuated these effects of S100A9, indicating that S100A9-induced microglia activation depends on TLR4 signaling. We examined whether or not the expression of TNF-α and IL-1β induced by S100A9 was inhibited by the treatment with PCF serum using the BV2 microglia. The expression of inflammatory markers was significantly upregulated by S100A9, and showed a downward trend by the co-treatment with PCF serum.
In the infarcted myocardium caused by prolonged coronary occlusion, the damage-associated molecular pattern (DAMP) proteins released from necrotic cells trigger both myocardial and systemic inflammatory responses. Inflammatory cells clear the infarct of dead cells and matrix debris, as well as activate repair by myofibroblasts and vascular cells, but may also lead to adverse fibrotic remodelling of viable segments and accentuate cardiomyocyte apoptosis. Induction of cytokines and up-regulation of endothelial adhesion molecules modulate leukocyte recruitment in the infarcted heart tissues. Apoptosis, a process of programmed cell death, has been proposed to occur in response to proinflammatory cytokines post myocardial ischemia. In the present study, we measured the inflammation level and occurrence of apoptosis severally by HE staining and TUNEL staining in the heart tissues on day 7 after coronary ligation. Also, Masson staining was applied to assess myocardial fibrosis at 21 days post-AMI. In the AMI group, a severe inflammatory infiltrates and myocardial fibre rupture were shown, as well as increased apoptosis index and fibrotic region. Conversely, these pathological phenomenons were alleviated by the administration of PCF.
There is considerable evidence that behavioural impairment observed after AMI are consistent with a model of human post-MI depression[51–53]. A majority of studies accorded closely with the conclusion, and the team of Wann put a lot of efforts to make it convinced[52, 53]. As reported by Wann, MI rats display behavioural signs compatible with depression 2 weeks after the cardiovascular event, including anhedonia (i.e., less sucrose intake) and behavioural despair (i.e., decreased forced swimming). Our study declared that rats in AMI group showed depression-like behavior, as performed by reduced ability of movement in OFT and longer immobility time in FST. These findings implied that depression-like performance in rodents with MI was demonstrated by diverse behavioral tests.
Depression is recognized as a circuit disease influencing multiple encephalic regions that are connected in functional networks. The hippocampus, as a primary zone in cerebral limbic system, has been identified as a major role in the pathological progress of depression. Many factors that may interact with hippocampal damage to trigger depressive episodes, and neurotransmitter disturbance and altered neurotrophic signaling are included. The 5-HT hypothesis of depressive disorder is supported by vast amounts of data that serotonin metabolism is altered in depression. The shunt of Try from 5-HT formation to Kyn formation is a dominating etiological factor of depression. Kyn was reported to be a proinflammatory metabolite in the neuroimmune signaling network mediating depressive-like behavior. The Kyn/Try ratio, an indicator of the activation of the first step of the Kyn pathway, the elevation of which indicated a decrease in the conversion of tryptophan to 5-HT. Activation of the Kyn pathway via inflammation has been substantiated both in clinical and preclinical research[57, 58]. Inflammation-driven alterations in kynurenine metabolic pathways results in substantial alterations in the metabolism of 5-HT. Our study showed a low content of 5-HT on day 7 after AMI, accompanied with a rise in Kyn/Try ratio. Myocardial infarction might disturb tryptophan metabolism through the kynurenine pathway, thereby resulting in a decrease in 5-HT synthesis. PCF might change the expression of 5-HT directly via the kynurenine pathway, thus improving depression-like behaviors in AMI rats.
One of the most attractive features of hippocampus is the unusual capacity for adult neurogenesis. In the sub-granular zone of the dentate gyrus (DG) of the hippocampus, new-born neurons are continuously generated, developed into mature neurons and functionally integrated into the existing neural circuitry. It’s now well established that adult hippocampal neurogenesis is decreased in rodent models of depression. Pro-inflammatory cytokines are involved in immune system-to-brain communication by activating resident microglia in the brain. Activated microglia reduce neurogenesis by suppressing neuronal stem cell proliferation, promoting apoptosis of neuronal progenitor cells, and decreasing survival of newly developing neurons and their integration into existing neuronal circuits. The process of neurogenesis is strongly stimulated by brain-derived neurotrophic factor (BDNF), a neurotrophic factor that modulates functional and structural plasticity in the central nervous system, thus affecting dendritic spines and adult neurogenesis. A mass of studies reported the association of a decrease in BDNF mRNA and protein levels in the hippocampus with an increase of susceptibility to develop depressive disorders[61, 62]. For synaptic plasticity, we observed the morphology and number of neurons in the hippocampus through Nissl staining. The experimental results showed that the neuronal body of the hippocampus in the AMI group is lost. In our study, the effects of the AMI model on the expression of synaptic-plasticity protein in the hippocampus were explored by western blotting. The decrease in BDNF might account in part for the depression-like behavior in AMI rats at 21 days postoperatively. The results were opposite for rats treated with PCF, although no significant statistical difference was found.
According to the theory of traditional Chinese medicine, blood stasis not only brings about obstructed meridian vessel, but also produces the stagnation of vital energy, thus characterized by chest pain and mental fatigue, respectively named as “Zhenxintong” (AMI) and “Yubing” (depression). PCF consists of four kinds of herbs, among which Salvia miltiorrhiza and Rhizoma chuanxiong could promote blood circulation to remove blood stasis, while Semen ziziphi spinosae and Lily bulbs could make the subjects vigorous and uplifting. There are some laboratory achievements for the molecule compounds of components in PCF consistent with the inflammatory mechanism obtained in our study. Our previous research suggested that PCF reduced the inflammatory reaction derived from myocardial ischemia, and changed the expression of neurotransmitters directly by modifying the expression of GAD67 to relieve depression. The published literature showed that the tanshinone IIA and salvianolate, as bioactive chemical constitutes from the root of Salvia miltiorrhiza, have significant advantage in cardiovascular protective effects, as well as the efficacy of anti-inflammatory and myocardial protective. Ferulic acid, an important active ingredient in Ligusticum chuanxiong, proved to be antidepressive via increasing monoamine neurotransmitter levels in the hippocampus.
There are limitations to our study. S100A9 is a small calcium-binding protein of the S100 family that is expressed, in most biological settings, as a heterodimer complexed with its partner, S100A8. Future researches concentrating on the functional and pathological difference between the monomer and heterodimer are urgently needed. Moreover, nothing but in vitro evidence was provided that S100A9 triggered microglial activation through TLR4 pathway, yet animal experiments in which a biological metabolism is more similar to the human body remains absent.