Phillyrin protects mice from traumatic brain injury by inhibiting the inflammation of microglia via PPARγ signaling pathway

Highlights

• Phillyrin attenuates TBI-mediated neurological deficits in mice.

• Phillyrin attenuates TBI-mediated microglial neuroinflammation.

• Phillyrin mediates microglia induced neuroinflammation through inhibiting NF-κB via activating PPARγ.

Abstract

The neuroinflammatory response induced by microglia plays a vital role in causing secondary brain damage after traumatic brain injury (TBI). Previous studies have found that the improved regulation of activated microglia could reduce neurological damage post-TBI. Phillyrin (Phi) is one of the main active ingredients extracted from the fruits of the medicinal plant Forsythia suspensa (Thunb.) with anti-inflammatory effects. Our study attempted to investigate the effects of phillyrin on microglial activation and neuron damage after TBI. The TBI model was applied to induce brain injury in mice, and neurological scores, brain water content, hematoxylin and eosin staining and Nissl staining were employed to determine the neuroprotective effects of phillyrin. Immunofluorescent staining and western blot analysis were used to detect nuclear factor-kappa B (NF-κB) and peroxisome proliferator–activated receptor gamma (PPARγ) expression and nuclear translocation, and the inflammation-related proteins and mRNAs were assessed by western blot analysis and quantitative real-time PCR. The results revealed that phillyrin not only inhibited the proinflammatory response induced by activated microglia but also attenuated neurological impairment and brain edema in vivo in a mouse TBI model. Additionally, phillyrin suppressed the phosphorylation of NF-κB in microglia after TBI insult. These effects of phillyrin were mostly abolished by the antagonist of PPARγ. Our results reveal that phillyrin could prominently inhibit the inflammation of microglia via the PPARγ signaling pathway, thus leading to potential neuroprotective treatment after traumatic brain injury.

Introduction

Traumatic brain injury (TBI) is one of the major causes of death and disability worldwide [1]. The primary impact of TBI is direct neural cell loss following a wave of secondary injury cascades, including excitotoxicity, oxidative stress, blood–brain barrier disruption and inflammation [2], [3], [4]. Studies have confirmed that overactive microglia play important roles in the cause of secondary injuries both during the early and late phases of TBI [5], [6]. As the immune cells of the central nervous system, microglia can be activated by molecules such as damage-associated molecular patterns (DAMPs) around the damage lesions [7], [8], [9]. Afterwards, superfluous inflammatory mediators including interleukin (IL)-1β, tumor necrosis factor-α (TNFα), and IL-6 are released from the activated microglia; those mediators could not only directly induce neuronal apoptosis and brain edema but also cause the destruction of the blood-brain barrier (BBB), thus further exacerbating CNS injury [10], [11], [12], [13]. Targeting the neuroinflammation mediated by microglia is thought to become an effective therapeutic strategy for TBI [14].

During the process of microglial activation, the toll-like receptors (TLRs), which are mainly expressed on the surfaces of microglia, respond rapidly to DAMPs. The downstream signaling pathways of TLRs, including myeloid differentiation factor 88 (MyD88) and NF-κB, are subsequently activated, accompanied by the upregulation of genes related to inflammation [15], [16]. Additionally, targeting TLRs or their downstream signaling pathways has shown conspicuous effects on regulating microglia-induced neuroinflammation [17], [18]. Drugs such as rosiglitazone [19] and pioglitazone [20], which can act as potent anti-inflammatory agents in the suppression of pro-inflammatory cytokine production, have shown prominent roles in decreasing the neurological deficits caused by TBI. The inhibition of posttraumatic neuroinflammation has been an important approach in the treatment of TBI.

Phillyrin (also called forsythin) is one of the main chemical constituents of Forsythia suspensa (Thunb.). In fact, as an important traditional Chinese medicine, F. suspensa (“Lianqiao” in Chinese) has historically been used for the treatment of influenza, inflammation, pyrexia and ulcers [21], [22], [23], [24]. Several studies have found that phillyrin can also exert anti-inflammatory effects against neutrophils in an LPS-induced inflammation model [25]. Specifically, phillyrin also exhibits protective effects on the central nervous system. For instance, Wei [26] found that phillyrin effectively inhibited H₂O₂-induced oxidative stress and apoptosis in PC12 cells. However, less is known about the effects of phillyrin on traumatic brain injury and posttraumatic neuroinflammation. In the present study, we attempted to study the anti-inflammatory effects of phillyrin on activated microglia as well as the neuroprotective effects in a mouse TBI model. Additionally, the potential mechanisms of phillyrin were also investigated.