Bone marrow mesenchymal stem cells therapy regulates sphingolipid and glycerophospholipid metabolism to promote neurological recovery in stroke rats: A metabolomics analysis.

Bone marrow mesenchymal stem cells (BMSCs) have therapeutic potential in the subacute/chronic phase of acute ischemic stroke (AIS), but the underlying mechanisms are not yet fully elucidated. There is a knowledge gap in understanding the metabolic mechanisms of BMSCs in stroke therapy. In this study, we administered BMSCs intravenously 24 h after reperfusion in rats with transient cerebral artery occlusion (MCAO). The treatment with BMSCs for 21 days significantly reduced the modified neurological severity score of MCAO rats (P < 0.01) and increased the number of surviving neurons in both the striatum and hippocampal dentate gyrus region (P < 0.01, respectively). Moreover, BMSCs treatment resulted in significant enhancements in various structural parameters of dendrites in layer V pyramidal neurons in the injured hemispheric motor cortex, including total length (P < 0.05), number of branches (P < 0.05), number of intersections (P < 0.01), and spine density (P < 0.05). Then, we performed plasma untargeted metabolomics analysis to study the metabolic changes of BMSCs on AIS. There were 65 differential metabolites identified in the BMSCs treatment group. Metabolic profiling analysis revealed that BMSCs modulate abnormal sphingolipid metabolism and glycerophospholipid metabolism, particularly affecting core members such as sphingomyelin (SM), ceramide (Cer) and sphingosine-1-phosphate (S1P). The metabolic network analysis and pathway-based compound-reaction-enzyme-gene network analysis showed that BMSCs inhibited the Cer-induced apoptotic pathway and promoted the S1P signaling pathway. These findings suggest that the enhanced effects of BMSCs on neuronal survival and synaptic plasticity after stroke may be mediated through these pathways. In conclusion, our study provides novel insight into the potential mechanisms of BMSCs treatment in stroke and sheds light on the possible clinical translation of BMSCs.

The "Dialogue" Between Central and Peripheral Immunity After Ischemic Stroke: Focus on Spleen.

The immune response generated by the body after the incidence of ischemic stroke, runs through the comprehensive process of aftermath. During this process of ischemic stroke, the central neuroinflammation and peripheral immune response seriously affect the prognosis of patients, which has been the focus of research in recent years. As this research scenario progressed, the "dialogue" between central nervous inflammation and peripheral immune response after ischemic stroke has become more closely related. It's worth noting that the spleen, as an important peripheral immune organ, plays a pivotal role in this dialogue. Multiple mechanisms have previously been reported for brain-spleen crosstalk after ischemic stroke. Further, neuroinflammation in the brain can affect the peripheral immune state by activating/inhibiting spleen function. However, the activation of the peripheral immune inflammatory response can work reversibly in the spleen. It further affects intracerebral neuroinflammation through the injured blood-brain barrier. Therefore, paying close attention to the role of spleen as the pivot between central and peripheral immunity in ischemic stroke may help to provide a new target for immune intervention in the treatment of ischemic stroke. In the present review, we reviewed the important role of spleen in central neuroinflammation and peripheral immune response after ischemic stroke. We summarized the relevant studies and reports on spleen as the target of immune intervention which can provide new ideas for the clinical treatment of ischemic stroke.

Bushen Kangshuai tablet inhibits progression of atherosclerosis by intervening in macrophage autophagy and polarization.

OBJECTIVE: To investigate the efficacy of Bushen Kangshuai (BS-KS) tablet on autophagy and polarization in mouse macrophage RAW 264.7. MEYHODS: Macrophage autophagy was induced by oxidized low-density lipoprotein (100 µg/mL). To detect the levels of autophagy, macrophage were transfected with double fluorescence LC3 autophagy adenovirus, then the numbers of autophagosomes and autophagic lysosomes were asessed by confocal microscopy. The autophagy related proteins expression of PI3K, Akt, phospho-mAkt (p-Akt) and mTOR, phospho-mTOR ([p-TOR), p62, microtubule-associated protein 1 (LC3-Ⅱ)were determined by western blotting. The macrophage polarization model was induced by lipopolysaccharide (1 µg/mL). The mRNA levels of iNOS, CD86 (M1 macrophages marker molecules), and CD206, Arg-1 (M2 macrophages marker molecules) were detected by real-time quantitative PCR. The concentration of cytokines TNF-α and IL-10 was determined by enzyme-linked immunosorbent assay. The protein expression of nuclear proteins PPAR-γ, NF-κB, and cytoplasmic protein IKB α was determined by western blotting. RESULTS: The expression of the autophagy-related protein LC3-Ⅱ was increased and the expression of p62 was decreased in the BS-KS intervention group. The protein expression of PI3K, p-Akt, and p-mTOR was also reduced. BS-KS also inhibited the mRNA expression of iNOS and CD86 on M2 macrophage, but promoted the expression of CD206 and Arg-1 on M2 macrophage. With respect to the regulation of inflammatory factors, BS-KS could inhibit the secretion of pro-inflammatory TNF-α and promote the secretion of anti-inflammatory IL-10. It also inhibited the protein expression of IKB-α and NF-κB, and promoted the expression of nuclear protein PPAR-γ. CONCLUSION: We believe that BS-KS promotes macrophage autophagy by increasing the level of autophagy protein and inhibiting the PI3K/Akt/mTOR signaling pathway. Furthermore, BS-KS seems to inhibit macrophage M1 polarization and promote M2 polarization via the PPAR gamma /NF-κB signaling pathway, thus playing an inhibitory role in atherosclerosis.

Neuroprotective effect of salvianolic acid B against cerebral ischemic injury in rats via the CD40/NF-κB pathway associated with suppression of platelets activation and neuroinflammation.

Neuroinflammation plays a critical role in the pathogenesis of ischemia/reperfusion (I/R) injury. Activated platelets are increasingly regarded as initiators and/or amplifiers of inflammatory processes in cerebral I/R injury. Salvianolic acid B (SAB) is the most abundant bioactive compound of Salviae miltiorrhizae, a well-known Chinese herb used to promote blood circulation and eliminating blood stasis. S. miltiorrhizae has been used clinically in Asia for the treatment of ischemic cerebrovascular diseases. In the present study, a rat model of transient middle cerebral artery occlusion (tMCAO) was established to investigate the neuroprotective effects and mechanisms of SAB treatment against focal cerebral I/R insult. The results showed that SAB treatment (3mg/kg, 6mg/kg and 12mg/kg, i.p.) dose-dependently decreased I/R-induced neurological deficits at 24, 48, and 72h after reperfusion and decreased plasma-soluble P-selectin and soluble CD40 ligand as early as 6h after onset of I/R insult. At 24h after reperfusion, SAB treatment significantly reduced neuronal and DNA damage in the hippocampal CA1 region and decreased neural cell loss in the ischemic core. The I/R-induced pro-inflammatory mediator mRNA and protein overexpression in the penumbra cortex, including ICAM-1, IL-1ß, IL-6, IL-8, and MCP-1, were significantly inhibited by SAB in a dose-dependent manner. Further studies suggested SAB treatment attenuated CD40 expression and NF-κB activation, which involved NF-κB/p65 phosphorylation and IκBα phosphorylation and degradation. In conclusion, our findings indicated that the neuroprotective effects of SAB post cerebral I/R injury are associated with the inhibition of both platelets activation and production of pro-inflammatory mediators and the downregulation of the CD40/NF-κB pathway.

Salvianolic acid B inhibits platelets-mediated inflammatory response in vascular endothelial cells.

Salvianolic acid B (SAB) is a hydrophilic component isolated from the Chinese herb Salviae miltiorrhizae, which has been used clinically for the treatment of ischemic cardiovascular and cerebrovascular diseases. Platelets-mediated vascular inflammatory response contributes to the initiation and progression of atherosclerosis. In this paper, we focus on the modulating effects of SAB on the inflammatory reaction of endothelial cells triggered by activated platelets. Human umbilical vein endothelial cells (EA.hy926) were pretreated with SAB followed by co-culture with ADP-activated platelets. Adhesion of platelets to endothelial cells was observed by amorphological method. The activation of nuclear factor-kappa B was evaluated by NF-κB p65 nuclear translocation and the protein phosphorylation. A determination of the pro-inflammatory mediators (ICAM-1, IL-1ß, IL-6, IL-8, MCP-1) mRNA and protein were also conducted. In addition, the inhibitory effects of SAB on platelets activation were also evaluated using a platelet aggregation assay and assessing the release level of soluble P-selectin. The results showed that SAB dose-dependently inhibited ADP- or α-thrombin-induced human platelets aggregation in platelet rich plasma (PRP) samples, and significantly decreased soluble P-selectin release from both agonists stimulated washed platelets. It was also found that pre-treatment with SAB reduced adhesion of ADP-activated platelets to EA.hy926 cells and inhibited NF-κB activation. In addition, SAB significantly suppressed pro-inflammatory mediators mRNA and protein in EA.hy926 cells in a dose-dependent manner. These results indicated that, in addition to its inhibitory effects on platelets activation, SAB was able to attenuate platelets-mediated inflammatory responses in endothelial cells even if the platelets had already been activated. This anti-inflammatory effect was related to the inhibition of NF-κB activation. Our findings suggest that SAB may be a potential candidate for the treatment of various atherosclerotic diseases.