Scutellarin regulates microglia-mediated TNC1 astrocytic reaction and astrogliosis in cerebral ischemia in the adult rats

Abstract

Background: Scutellarin, an anti-inflammatory agent, effectively suppressed microglia activation in rats with middle cerebral artery occlusion (MCAO). Robust microglia activation, acute in onset, was followed by astrogliosis. This study was aimed to determine if scutellarin would also affect the reactive astrocytes that play an important role in tissue repair. Expression of GFAP and Notch-1 and its members: Notch receptor intracellular domain (NICD), and transcription factor hairy and enhancer of split-1 (HES-1), together with nestin and proinflammatory mediators was assessed by immunofluorescence staining in TNC1 astrocytes treated, respectively, with BV-2 conditioned medium (CM) and CM + lipopolysaccharide (LPS) (CM + L) serving as the controls, and conditioned medium derived from LPS-activated BV-2 cells pretreated with scutellarin (CM + SL). Study of the above biomarkers was then extended to reactive astrocytes in scutellarin injected MCAO rats.

Results: TNC1 astrocytes remained relatively unreactive in terms of expression of different biomarkers to direct scutellarin treatment when compared with the control cells. In comparison to cells in the control medium (CM, CM + L), they responded vigorously to CM + SL as evidenced by the enhanced protein expression of GFAP, Notch-1, NICD and HES-1 coupled with that of nestin, TNF-α, IL-1β, and iNOS by Western and immunofluorescence analysis. Electron microscopy showed marked hypertrophy and cell expansion of TNC1 astrocytes bearing many filamentous processes indicative of enhanced astrocyte reaction when treated with CM + SL. In MCAO rats, scutellarin also augmented the expression of the above markers in reactive astrocytes; moreover, astrocytes were evidently hypertrophic.

Conclusions: The results suggest that scutellarin regulates astrogliosis; more importantly, it is microglia-mediated as demonstrated in vitro. Increased expression of Notch signaling in synchrony with nestin may be linked to proliferation and "de-differentiation" of reactive astrocytes; the significance of enhanced TNF-α, IL-1β and iNOS expression in reactive astrocytes by scutellarin may be neuroprotective but this remains speculative.

Fig. 1

Cell viability assay of TNC1 astrocytes (a): scutellarin (in the range of 0.2–2.0 mM) incubated for different duration did not result in any significant cell death. Treatment of TNC1 with scutellarin (b): scutellarin at 0.54 mM did not elicit a noticeable reaction in TNC1 whose GFAP/TNF-α (B1–3) expression remained comparable to cells in the control in basic medium (BM) (A1–3). BM + S, basic medium + scutellarin. Microglia mediate TNC1 astrocyte reaction (c): TNF-α mRNA expression in TNC1 astrocytes remained relatively unchanged at all time-points following treatment with BM, BM + L and BV-2 conditioned medium (CM). However, in TNC1 incubated CM + L for various time points, TNC1 showed a remarkable increase in TNF-α peaking at 24 h. Confocal images in d showing GFAP (C1–3), and TNF-α (D1–3) expression in TNC1 astrocytes incubated with different medium for 24 h. Compared with cells incubated in BM and BM + LPS (BM + L), TNC1 astrocytes incubated with LPS-stimulated BV-2 cell conditioned medium (CM + L) (C3, D3) are hypertrophic and exhibit a marked increase in GFAP and TNF-α immunofluorescence. Scale bars 20 μm

Fig. 2

Scutellarin enhanced GFAP (A1–A3), Notch-1 (B1–B3), NICD (C1–C3), HES-1 (D1–D3), nestin (E1–E3) and TNF-α (A1–3) expression in TNC1 via BV-2-conditioned medium. Moderate GFAP expression was detected in TNC1 incubated in CM; also, Notch-1, NICD, HES-1, nestin and TNF-α (B1, C1, D1, E1, F1) was weakly expressed. The expression however was enhanced in CM + L (B2, C2, D2, E2, F2). Upon treatment with CM + SL for 24 h, expression of all markers was drastically increased being more pronounced for NICD (C3) and nestin (E3). Note TNC1 astrocytes project long cytoplasmic processes with expansions (A3, D3, F3). Scale bars 20 μm

Fig. 3

Protein expression of GFAP, Notch-1, NICD, HES-1, TNF-α, IL-1β and iNOS in TNC1 following different treatments. a Expression levels of GFAP, Notch-1, NICD, and HES-1 were significantly increased after treatment with CM + L when compared with the control in CM; likewise, the expression of TNF-α, IL-1β and iNOS was significantly increased. b In CM + SL treated TNC1, the expression levels of the above markers were further elevated as compared with that of CM + L treated cells. Bar graphs represent expression changes of the respective markers. Significant differences in protein levels are expressed as *^#P < 0.05. The values represent the mean ± SD in triplicate. ^#CM + L vs CM, *CM + SL vs CM + L

Fig. 4

Scanning electron microscopy of TNC1 astrocytes in CM (A1–2), CM + SL (B1–2) and CM + SL (C1–2) groups. Note the drastic transformation of TNC1 from oblong outline (CM, A1–2) to “squamous” appearance in the CM + SL (C1–2) whose cell surface exhibit a large number of filamentous processes (arrows). By transmission electron microscopy (D1–3), TNC1 astrocytes in CM + SL are evidently enlarged (D3) as compared with CM (D1) and CM + L (D2) groups; moreover, the cells contain a larger amount of cytoplasm rich in polyribosomes and usual organelles

Fig. 5

Morphological evidence of interaction between activated microglia and reactive astrocytes. Figure showing ipsilateral ischemic cerebral cortex at 3 days after MCAO. The dotted line in A delineates approximately the border of the infarct epicenter which shows massive accumulation of activated microglia intermingled with reactive astrocytes. B1, B2 show lectin-labeled activated microglia (green). They are obviously hypertrophic and are mostly rounded (B1) or emit short stout processes (B2). GFAP labeled reactive astrocytes (red) are also hypertrophic, and extend their long processes between the activated microglia; in some areas, they are closely associated (B1, B2). Astrocytic processes also surround the blood vessel (BV) in B1, B2. The close spatial relation between the activated microglia and reactive astrocytes is not evident away from the border (B3). B1–3 are enlarged view of area 1–3 in A. Scale bar in A: 100 μm; scale bar in B: 50 μm. DAPI, blue

Fig. 6

Scutellarin enhanced Notch-1expression in astrocytes after MCAO. Notch-1 expression is negligible in astrocytes in the sham (A1–3). Its expression (red) was moderately induced by MCAO in GFAP positive astrocytes (green) at 7 (B1–3) and 14 days (D1–3). However, Notch-1 expression was markedly increased in hypertrophic astrocytes in scutellarin treated MCAO rats (C1–3, E1–3) in comparison with the matching controls (B1–3, D1–3). Scale bars: 50 µm. DAPI-blue

Fig. 7

Scutellarin enhanced NICD expression in astrocytes after MCAO. NICD expression is absent in astrocytes in the sham (A1–3). Its expression (red) was moderately induced after MCAO in GFAP positive astrocytes (green) at 7 days (B1–3) but was diminished at 14 days (D1–3). However, NICD expression was markedly increased in hypertrophic astrocytes in scutellarin treated MCAO rats (C1–3, E1–3) in comparison to the matching control groups (B1–3, D1–3). Scale bars: 50 µm. DAPI-blue

Fig. 8

Scutellarin enhanced HES-1 expression in astrocytes after MCAO. HES-1 expression is undetected in astrocytes in the sham (A1–3). Its expression (red) was moderately induced after MCAO in GFAP positive astrocytes (green) at 7 days (B1–3) but was attenuated at 14 days (D1–3). However, HES-1 expression was markedly increased in hypertrophic astrocytes in scutellarin treated MCAO rats at 7 (C1–3) and 14 days (E1–3) in comparison to the matching control groups (B1–3, D1–3). Scale bars: 50 µm. DAPI-blue

Fig. 9

Scutellarin enhanced nestin expression in astrocytes after MCAO. Nestin expression is undetected in astrocytes in the sham (A1–3). It (red) was moderately induced in GFAP positive astrocytes (green) in MCAO rats at 7 (B1–3) but declined at 14 days (D1–3). However, nestin expression was markedly increased in hypertrophic astrocytes in scutellarin treated MCAO rats at 7 (C1–3) and 14 days (E1–3) in comparison to the matching control groups (B1–3, D1–3). Scale bars: 50 µm. DAPI-blue

Fig. 10

Scutellarin enhanced TNF-α expression in astrocytes after MCAO. TNF-α expression (red) was absent in astrocytes in the sham (A1–3). It was induced in GFAP positive astrocytes (green) at 3 and 7 days (B1–3) after MCAO, but the expression subsided at 14 days (D1–3). In MCAO rats treated with scutellarin (C1–3, E1–3). TNF-á expression in astrocytes was markedly enhanced being most pronounced at 7 days when compared with the matching MCAO control groups (B1–3, D1–3). Scale bars: 50 µm. DAPI-blue