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Silybin attenuates microglia-mediated neuroinflammation via inhibition of STING in experimental subarachnoid
Yue Cui1, Si-Min Zhi1, Peng-Fei Ding2
1Department of Neurosurgery, Nanjing Drum Tower Hospital Clinical College of Nanjing University of Chinese Medicine, Nanjing, China; Neurosurgical Institute, Nanjing University.
Background:
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.
Purpose:
To determine the therapeutic effect of silybin on SAH and its underlying mechanisms.
Methods:
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:
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.
Conclusion:
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.
Insights
Silybin, a natural compound, shows therapeutic potential for subarachnoid hemorrhage (SAH) by reducing brain injury and inflammation. It acts by modulating microglial polarization and the STING pathway, offering a new treatment strategy for SAH patients.
Area of Science:
- Neuroscience
- Pharmacology
- Biochemistry
Background:
- Subarachnoid hemorrhage (SAH) is primarily caused by ruptured intracranial aneurysms, leading to early brain injury (EBI) driven by microglial over-activation.
- Silybin, a flavonoid from Silybum marianum, possesses anti-inflammatory and antioxidant properties with potential neuroprotective effects, but its mechanism in SAH remains unclear.
Purpose of the Study:
- To investigate the therapeutic efficacy of silybin in treating SAH.
- To elucidate the underlying molecular mechanisms of silybin's neuroprotective effects in SAH.
Main Methods:
- Established experimental SAH models in vivo and in vitro using arterial blood injection and hemoglobin.
- Assessed silybin's neuroprotective effects via neurological function tests and analyzed neuroinflammation and apoptosis using qPCR, Western blot, immunofluorescence, and TUNEL staining.
- Investigated microglial polarization, the STING pathway, and employed transcriptomics and metabolomics to uncover detailed mechanisms, validated in SAH patient cerebrospinal fluid.
Main Results:
- Silybin significantly improved neurological function, reduced neuronal damage, inflammation, and apoptosis in SAH mice.
- Silybin promoted M2 microglia polarization and inhibited the STING pathway, reducing pro-inflammatory markers (TNF-α, IL-1β, NLRP3) and increasing anti-inflammatory markers (CD206, Arg1, IL-10).
- Transcriptomic and metabolomic analyses identified linoleic acid metabolism as a key pathway affected by STING, with STING levels in patient CSF correlating with neurological function and prognosis.
Conclusions:
- Silybin demonstrates significant therapeutic effects against SAH, offering neuroprotection through mechanisms involving microglial polarization and the STING pathway.
- The study highlights the role of linoleic acid metabolism in silybin's action and provides a strong pharmacological basis for its use in treating SAH.
