Alpinetin inhibits neuroinflammation and neuronal apoptosis via targeting the JAK2/STAT3 signaling pathway in spinal cord injury

Abstract

Background: A growing body of research shows that drug monomers from traditional Chinese herbal medicines have antineuroinflammatory and neuroprotective effects that can significantly improve the recovery of motor function after spinal cord injury (SCI). Here, we explore the role and molecular mechanisms of Alpinetin on activating microglia-mediated neuroinflammation and neuronal apoptosis after SCI.

Methods: Stimulation of microglia with lipopolysaccharide (LPS) to simulate neuroinflammation models in vitro, the effect of Alpinetin on the release of pro-inflammatory mediators in LPS-induced microglia and its mechanism were detected. In addition, a co-culture system of microglia and neuronal cells was constructed to assess the effect of Alpinetin on activating microglia-mediated neuronal apoptosis. Finally, rat spinal cord injury models were used to study the effects on inflammation, neuronal apoptosis, axonal regeneration, and motor function recovery in Alpinetin.

Results: Alpinetin inhibits microglia-mediated neuroinflammation and activity of the JAK2/STAT3 pathway. Alpinetin can also reverse activated microglia-mediated reactive oxygen species (ROS) production and decrease of mitochondrial membrane potential (MMP) in PC12 neuronal cells. In addition, in vivo Alpinetin significantly inhibits the inflammatory response and neuronal apoptosis, improves axonal regeneration, and recovery of motor function.

Conclusion: Alpinetin can be used to treat neurodegenerative diseases and is a novel drug candidate for the treatment of microglia-mediated neuroinflammation.

Keywords: Alpinetin; JAK2/STAT3 pathway; neurinflammation; neuronal apoptosis; spinal cord injury.

FIGURE 1

Lipopolysaccharide (LPS) triggers microglial activation in a time‐dependent manner. (A–C) Western blot images and quantification showing iNOS and COX‐2 protein levels in microglia. (D, E) Immunolabeling and quantification of Iba‐1 intensity in microglia. (F, G) Changes in the morphology of microglia with or without LPS stimulation. N = 3 per group for Western blot assay, N = 3 per group for Immunofluorescence staining assay. *p < 0.05 and **p < 0.01.

FIGURE 2

Alpinetin inhibits the release of proinflammatory mediators in lipopolysaccharide‐activated microglia. (A) The chemical structure of Alpinetin. (B) CCK8 assay of Alpinetin on BV2 viability for 24 h (control group is treated without Alpinetin). (C) Morphological changes in microglia. (D–G) The expressions of iNOS, COX‐2, TNF‐α and IL‐1β were assessed by RT‐qPCR. (H, I) Immunofluorescence staining and quantitative data revealed the expression levels of CD11b and Iba‐1 in microglia. (G–L) Western blot images and quantification showing iNOS and COX‐2 protein levels in microglia. N = 3 per group for Western blot assay, N = 3 per group for Immunofluorescence staining assay. *p < 0.05, **p < 0.01 and ***p < 0.001.

FIGURE 3

Alpinetin inhibit lipopolysaccharide‐mediated neuroinflammatory response in microglia via targeting JAK2/STAT3 signaling pathway. (A) Molecular docking of Alpinetin molecule and JAK2 protein was performed using Autodock vina. (B) Space filling model showing Alpinetin binding in the JAK2 binding pocket. (C–H) Western blot images and quantification showing p‐JAK2, JAK2, p‐STAT3, and STAT3 in microglia. (I–K) Western blot protein expressions and quantification data of iNOS and COX‐2. (L) The intracellular localization of p‐STAT3 (red fluorescence) was detected by immunofluorescence. N = 3 per group for Western blot assay, N = 3 per group for Immunofluorescence staining assay. *p < 0.05 and **p < 0.01.

FIGURE 4

Neuroprotection of APT against activated microglia‐mediated neurotoxicity. (A) Schematic diagram of co‐culture of BV2 microglia and PC12 cells. (B, C) Immunofluorescence staining and quantitative data revealed the expression levels of Bcl‐xL (red) and phalloidin(green) in each group of PC12 cells. (D, E) Western blot images and quantification showing Bcl‐xL protein levels in PC12 cells. (F) Changes in MMPs in PC12 neurons were captured by fluorescence microscopy. (G) Quantitative data analyzed the red/green ratio of PC12 cells. (H) Intracellular ROS detection with dichloro‐dihydro‐fluorescein diacetate (DCFH‐DA) obtained by immunofluorescence staining. (I) Quantification of the proportion of ROS‐positive PC12 cells. N = 3 per group for Western blot assay, N = 3 per group for Immunofluorescence staining assay. *p < 0.05 and **p < 0.01.

FIGURE 5

Alpinetin improves the recovery of motor function in rats after spinal cord injury. (A) Basso‐Beattie‐Bresnahan (BBB) scores of different groups of rats at different time points after injury. (B–E) Quantitative analysis of the BBB score at 14, 28, 42, and 56 days after injury. (F) Footprint analysis to assess hindlimb motor function recovery. Forelimb footprints are shown in red and hindlimb footprints are shown in blue. (G, H) Quantification of stride length and base of support to assess motor recovery at 28 and 56 days postinjury. (I, J) Image and quantification of H&E‐stained cavity areas in longitudinal and transverse sections at 56 days postinjury. (K) The electrophysiology of each group was detected. (L, M) Quantitative analysis of amplitudes of motor evoked potential in each group. N ≥ 9 per group for BBB score, N = 4 per group for footprint assay, N = 3 per group for H&E staining of longitudinal and transverse sections at 56 days postinjury. *p < 0.05 and **p < 0.01.

FIGURE 6

Alpinetin inhibits inflammatory response after spinal cord injury. (A, B) Image and quantification of Iba‐1 (green)/and GFAP (red) staining in longitudinal sections of spinal cords of rats in each group at 3 days postinjury. (C, D) Image and quantification of Iba‐1 (green)/and GFAP (red) staining in transverse sections of spinal cords of rats in each group at 3 days postinjury. (E–I) iNOS, COX‐2 and Iba‐1 proteins were detected and quantified in each group at 3 days postinjury. N = 3 per group for histology analysis, N = 3 per group for Western blot assay. *p < 0.05 and **p < 0.01.

FIGURE 7

Alpinetin attenuates neuronal apoptosis after spinal cord injury. (A) Neuronal survival in transverse section on Day 3 postinjury was assessed by Nissl staining. (B, C) NeuN protein level were detected and quantified in each group at 3 days postinjury. (D, E) At 3 days after injury, the expression of antiapoptosis‐related protein Bcl‐xL was detected and quantified in each group. (F) Double immunofluorescence staining labeled NeuN (green) and Bcl‐xL (red) on longitudinally sectioned tissue in each group at 3 days postinjury. (G) Quantitative analysis of the fluorescence intensity of Bcl‐xL in neurons. N = 3 per group for histology analysis, N = 3 per group for Western blot assay. *p < 0.05 and **p < 0.01.

FIGURE 8

Alpinetin promotes axonal regeneration after spinal cord injury. (A) Double immunofluorescence staining showed GFAP (green) and MAP‐2 (red) in each group at 56 days postinjury. The vertical white dotted line represents the center of the lesion, and the horizontal dotted line length represents the distance from the injury center to the nearest neuron in the caudal. (B) Quantification of the distance from the neuron to the center of the lesion. (C) Quantifying the positive intensity of MAP‐2 from A. (D) Double immunofluorescence staining showed GFAP (green) and GAP43 (red) in each group at 56 days postinjury. (E) Quantification of GAP43 fluorescence intensity in the lesion area from D. (F–H) Western blot images and quantification showing MAP‐2 and GAP43 protein levels in the lesion area. N = 3 per group for histology analysis, N = 3 per group for Western blot assay. *p < 0.05 and **p < 0.01.

FIGURE 9

Alpinetin's therapeutic effects on spinal cord injury (SCI) are depicted in this diagram. Alpinetin alleviates the inflammatory response and neuronal toxicity caused by activated microglia via targeting the JAK2/STAT3 pathway, and ultimately promotes functional recovery in SCI rats.