Silybin attenuates microglia-mediated neuroinflammation via inhibition of STING in experimental subarachnoid hemorrhage.

The primary cause of subarachnoid hemorrhage (SAH) is the rupture of intracranial aneurysms. Over-activation of microglia following SAH is a primary driving force in early brain injury (EBI), which is a leading cause of poor outcomes. Silybin is a flavonoid compound extracted from Silybum marianum, a plant belonging to the Asteraceae family. Its anti-inflammatory and antioxidant properties could provide neuroprotective effects. The mechanism of silybin on EBI after SAH is unclear. To determine the therapeutic effect of silybin on SAH and its underlying mechanisms. We used a prechiasmatic autologous arterial blood injection in vivo and hemoglobin in vitro to establish experimental SAH model. Dexamethasone was used as a positive control drug. We evaluated the neuroprotective effect of silybin on the in vivo SAH model by neurological function scores, rotarod test, and open field test, and explored the protective effect of silybin on neuroinflammation and apoptosis after SAH by quantitative polymerase chain reaction (qPCR), western blot (WB), Immunofluorescence (IF) and TUNEL staining. IF staining of CD86 and CD206 was used to assess microglial phenotype polarization. Then we used WB and IF labeling of STING to explore the effect of silybin on the STING pathway after SAH, and used a combination of transcriptomics and non-targeted metabolomics to study the potential mechanism of silybin in detail, and verified the essential genes by qPCR. We also extracted cerebrospinal fluid from SAH patients and detected the expression level of STING in cerebrospinal fluid by enzyme-linked immunosorbent assay (ELISA) to clarify the association between STING and neural function. Results showed that silybin ameliorated neuronal damage and improved short-term neurological function, and reduced inflammatory damage and neuronal apoptosis in SAH mice. Silybin inhibited the expression levels of TNF-α, IL-1β and NLRP3, and promoted the expression levels of CD206, Arg1 and IL-10. Notably, Silybin promoted M2 microglia polarization. Further studies found that silybin reduced the mRNA and protein levels of the stimulator of interferon genes (STING) in microglia. And the use of a specific activator of STING (CMA) disrupted the protective effect of silybin. A total of 358 differential expression genes were identified using transcriptomics, and 150 different metabolites abundance were identified using metabolomic screening. Analysis of the effects of STING on transcriptomics and metabolomics revealed that STING might impact metabolic pathways, including linoleic acid metabolism. The qPCR results confirmed the decreased expression of essential proteins involved in the pathway. Finally, we found that increased STING expression in the cerebrospinal fluid of SAH patients was associated with decreased neurological function scores and poor prognosis. Silybin had a therapeutic effect on SAH. The underlying mechanism involves linoleic acid metabolism, which is associated with the differential genes and metabolites detected in the study. This study presented a pharmacological rationale for using silybin to treat SAH.

Cui Y ，Zhi SM ，Ding PF ，Zhu T ，Chen XX ，Liu XZ ，Sheng B ，Li XJ ，Wang J ，Zhang JT ，Xu MX ，Jiang YX ，Hang CH ，Li W ... -  《-》

Danhong injection modulates microglial polarization and neuroinflammation via the JUNB/NF-κB pathway in ischemic stroke.

Ischemic stroke (IS) is a leading cause of death and disability in China. Danhong Injection (DHI) is a traditional Chinese medicine preparation made from Salvia miltiorrhiza var. miltiorrhiza and Carthamus tinctoriusL. It is used for treating stroke in China with proven safety and efficacy. Microglia M1/M2 polarization is a key factor in IS inflammatory response. However, the key transcription factors that regulate microglia polarization are unknown. It is also not clear how DHI exerts its mechanism in the treatment of IS. This research aimed to investigate the effect of DHI on microglial polarization and neuroinflammation associated with IS and to elucidate the underlying mechanisms, with an emphasis on the JUNB/NF-κB signaling pathway. An oxygen-glucose deprivation (OGD) damage cell model and a permanent middle cerebral artery occlusion (pMCAO) model in C57BL/6 mice were employed. Neurological deficits, cerebral infarct volume, and microglial activation were assessed. Non-targeted metabolomics analysis with UHPLC-QE-MS and molecular biology methods, including RT-qPCR and Western blot, were applied to investigate the mechanisms. In vivo, DHI decreased inflammation, reduced brain damage, and enhanced neurological function. DHI also ameliorated microglial activation and OGD-induced apoptosis in vitro. Metabolomics analysis identified significant metabolic changes, particularly in amino acid metabolism. Additionally, DHI treatment decreased the upregulated mRNA levels of ASS1 and ASL after OGD, indicating an influence on the arginine biosynthesis pathway, which is crucial for microglial function. DHI modulated the M1 to M2 phenotypes of microglial polarization and regulated microglial polarization through the JUNB/NF-κB signaling pathway. This was confirmed by JUNB silencing experiments. DHI exhibits neuroprotective effects via suppressing ASS1 through the JUNB/NF-κB pathway, promoting the M2 state of microglia, and lowering the expression of inflammatory cytokines. This research unveils the potential therapeutic target of JUNB for IS treatment and sheds light on the novel intervention mechanism of DHI in microglial cells.

Xie M ，Huang H ，Lu Y ，Chen L ，Wang S ，Xian M ... -  《-》

Hypothyroidism Promotes Microglia M1 Polarization by Inhibiting BDNF-Promoted PI3K-Akt Signaling Pathway.

Hypothyroidism and its induced neurological-associated disorders greatly affect the health-related quality of patients' life. Meanwhile, microglia in brain have essential regulatory functions on neurodegeneration, but the underlying link between hypothyroidism and microglia function is largely ambiguous. We deciphered how hypothyroidism modulates the polarization of microglia by constructing methimazole-induced mice model and checking the expression pattern of biomarkers of microglia M1 polarization. Then, we used lipopolysaccharide (LPS)-treated BV2 cells to explore the effecting factors on microglia M1 polarization. Finally, global transcriptome sequencing (RNA-seq) was utilized to identify the underlying regulatory mechanisms. We detected that biomarkers of microglia M1 polarization and pro-inflammatory cytokines were significantly increased in hypothyroidism mice brain; hypothyroidism could also repress the expression of BDNF and TrkB, and the anti-inflammatory cytokine such as IL-10. In BV2 cells, LPS treatment decreased expression of BDNF, IL-10, and Arg1, while BDNF overexpression (BDNF-OE) significantly reversed the inflammation induced by LPS. BDNF-OE significantly repressed expression of iNOS and TNF-α, but increased expression of IL-10 and Arg1. For mechanism, RNA-seq analysis demonstrated that BDNF-OE could globally regulate transcriptome profile by affecting gene expression. In LPS-treated BV2 cells, BDNF-OE significantly altered expression pattern of genes involved in PI3K-Akt signaling pathway, including Thbs3, Myc, Gdnf, Thbs1, and Ccnd1 as upregulated genes, and Gnb4, Fgf22, Pik3r3, Pgf, Cdkn1a, and Pdgfra as downregulated genes. Myc, Gdnf, Thbs1, and Ccnd1 showed much higher expression levels than other genes in PI3K-Akt signaling pathway and could be promising targets of BDNF in reversing microglia M1 polarization. Our study demonstrated a sound conclusion that hypothyroidism promotes microglia M1 polarization by inhibiting BDNF expression in brain; BDNF could inhibit the M1 polarization of microglia by activating PI3K-Akt signaling pathway, which could serve as a promising therapeutic target for microglia-induced neurodegenerative or emotional disorders in future. Hypothyroidism and its induced neurological-associated disorders greatly affect the health-related quality of patients' life. Meanwhile, microglia in brain have essential regulatory functions on neurodegeneration, but the underlying link between hypothyroidism and microglia function is largely ambiguous. We deciphered how hypothyroidism modulates the polarization of microglia by constructing methimazole-induced mice model and checking the expression pattern of biomarkers of microglia M1 polarization. Then, we used lipopolysaccharide (LPS)-treated BV2 cells to explore the effecting factors on microglia M1 polarization. Finally, global transcriptome sequencing (RNA-seq) was utilized to identify the underlying regulatory mechanisms. We detected that biomarkers of microglia M1 polarization and pro-inflammatory cytokines were significantly increased in hypothyroidism mice brain; hypothyroidism could also repress the expression of BDNF and TrkB, and the anti-inflammatory cytokine such as IL-10. In BV2 cells, LPS treatment decreased expression of BDNF, IL-10, and Arg1, while BDNF overexpression (BDNF-OE) significantly reversed the inflammation induced by LPS. BDNF-OE significantly repressed expression of iNOS and TNF-α, but increased expression of IL-10 and Arg1. For mechanism, RNA-seq analysis demonstrated that BDNF-OE could globally regulate transcriptome profile by affecting gene expression. In LPS-treated BV2 cells, BDNF-OE significantly altered expression pattern of genes involved in PI3K-Akt signaling pathway, including Thbs3, Myc, Gdnf, Thbs1, and Ccnd1 as upregulated genes, and Gnb4, Fgf22, Pik3r3, Pgf, Cdkn1a, and Pdgfra as downregulated genes. Myc, Gdnf, Thbs1, and Ccnd1 showed much higher expression levels than other genes in PI3K-Akt signaling pathway and could be promising targets of BDNF in reversing microglia M1 polarization. Our study demonstrated a sound conclusion that hypothyroidism promotes microglia M1 polarization by inhibiting BDNF expression in brain; BDNF could inhibit the M1 polarization of microglia by activating PI3K-Akt signaling pathway, which could serve as a promising therapeutic target for microglia-induced neurodegenerative or emotional disorders in future.

Zhan Y ，Lang L ，Wang F ，Wu X ，Zhang H ，Dong Y ，Yang H ，Zhu D ... -  《-》

Protective role of mitophagy on microglia-mediated neuroinflammatory injury through mtDNA-STING signaling in manganese-induced parkinsonism.

Manganese (Mn), the third most abundant transition metal in the earth's crust, has widespread applications in the emerging field of organometallic catalysis and traditional industries. Excessive Mn exposure causes neurological syndrome resembling Parkinson's disease (PD). The pathogenesis of PD is thought to involve microglia-mediated neuroinflammatory injury, with mitochondrial dysfunction playing a role in aberrant microglial activation. In the early stages of PD, PINK1/Parkin-mediated mitophagy contributes to the microglial inflammatory response via the cGAS/STING signaling pathway. Suppression of PINK1/Parkin-mediated mitophagy due to excessive Mn exposure exacerbates neuronal injury. Moreover, excessive Mn exposure leads to neuroinflammatory damage via the microglial cGAS-STING pathway. However, the precise role of microglial mitophagy in modulating neuroinflammation in Mn-induced parkinsonism and its underlying molecular mechanism remains unclear. Here, we observed that Mn-exposed mice exhibited neurobehavioral abnormalities and detrimental microglial activation, along with increased apoptosis of nerve cells, proinflammatory cytokines, and intracellular ROS. Furthermore, in vivo and in vitro experiments showed that excessive Mn exposure resulted in microglial mitochondrial dysfunction, manifested by increased mitochondrial ROS, decreased mitochondrial mass, and membrane potential. Additionally, with the escalating Mn dose, PINK1/Parkin-mediated mitophagy changed from activation to suppression. This was evidenced by decreased levels of LC3-II, PINK1, p-Parkin/Parkin, and increased levels of p62 protein expression level, as well as the colocalization between ATPB and LC3B due to excessive Mn exposure. Upregulation of mitophagy by urolithin A could mitigate Mn-induced mitochondrial dysfunction, as indicated by decreased mitochondrial ROS, increased mitochondrial mass, and membrane potential, along with improvements in neurobehavioral deficits and attenuated detrimental microglial activation. Using single-nucleus RNA-sequencing (snRNA-seq) analysis in the Mn-exposed mouse model, we identified the microglial cGAS-STING signaling pathway as a potential mechanism underlying Mn-induced neuroinflammation. This pathway is associated with an increase in cytosolic mtDNA levels, which activate STING signaling. These findings point to the induction of microglial mitophagy as a viable strategy to alleviate Mn-induced neuroinflammation through mtDNA-STING signaling.

Lu Y ，Gao L ，Yang Y ，Shi D ，Zhang Z ，Wang X ，Huang Y ，Wu J ，Meng J ，Li H ，Yan D ... -  《Journal of Neuroinflammation》

Curcumin Alleviates Microglia-Mediated Neuroinflammation and Neuronal Ferroptosis Following Experimental Subarachnoid Hemorrhage by Modulating the Nrf2/HO-1 Signaling Pathway.

Early brain injury caused by subarachnoid hemorrhage (SAH) is associated with inflammatory response and ferroptosis. Curcumin alleviates neuroinflammation and oxidative stress by as yet unknown neuroprotective mechanisms. The objective of this study was to investigate the impact of curcumin on neuronal ferroptosis and microglia-induced neuroinflammation following SAH. By examining Nrf2/HO-1 expression levels and ferroptosis biomarkers expression both in vitro and in vivo, it was demonstrated that curcumin effectively suppressed ferroptosis in neurons after SAH through modulation of the Nrf2/HO-1 signaling pathway. Furthermore, by analyzing the expression levels of Nrf2, HO-1, p-p65, and inflammation-related genes, it was confirmed that curcumin could prevent the upregulation of pro-inflammatory factors following SAH by regulating the Nrf2/HO-1/NF-κB signaling pathway in microglia. The ability of curcumin to reduce neuronal damage and cerebral edemas after SAH in mice was validated using TUNEL staining, Nissl staining, and measurement of brain tissue water content. Additionally, through implementation of the modified Garcia test, open field test, and Y-maze test, it was established that curcumin ameliorated neurobehavioral impairments in mice post-SAH. Taken together, these data suggest that curcumin may offer a promising therapeutic approach for improving outcomes following SAH by concurrently attenuating neuronal ferroptosis and reducing neuroinflammation.

Xu Y ，Liu Y ，Wu Y ，Sun J ，Lu X ，Dai K ，Zhang Y ，Luo C ，Zhang J ... -  《-》