Compound 21, a Direct AT2R Agonist, Induces IL-10 and Inhibits Inflammation in Mice Following Traumatic Brain Injury

Abstract

Recent studies demonstrated that the angiotensin type 2 receptor (AT2R) agonist, compound 21 (C21), provides neuroprotection and enhances recovery in experimental stroke. However, C21 has never been tested in traumatic brain injury (TBI). Here, we aim to examine whether C21 confers protection after TBI. Unilateral cortical impact injury was induced in young adult C57BL/6 mice. C21 (0.03 mg/kg, i.p.) was administered at 1 h and 3 h post-TBI. After neurological severity score (NSS) assessments, all animals were sacrificed for immunoblotting analysis at 24 h post-TBI. C21 treatment significantly ameliorated NSS and reduced TBI's biomarkers [high mobility group box 1 (HMGB1), aquaporin-4 (AQ4)] and inflammatory markers [interlukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α)] in the pericontusional areas compared to saline TBI. Further, C21 treatment induced interleukin-10 (IL-10) and phosphorylation of endothelial nitric oxide synthase (eNOS) after TBI. C21 also attenuated pro-apoptotic activation of poly (ADP-ribose) polymerase (PARP) and caspase-3. These findings support the therapeutic potential of C21 against TBI.

Keywords: AQ4; AT2R; Compound 21; Inflammation; TBI.

Fig. 1

Effect of C21 on pathological molecular markers after TBI. A Representative and B–I quantitative analysis of Western blots showed the differential expression of key pathological markers of TBI. B Expression of AQ4 is increased significantly at 24 h in saline-treated TBI mice compared to shams. C21 treatment significantly decreased AQ4 expression. C Quantitative analysis showed an elevation in HMGB1 expression in saline-treated TBI mice compared to shams. C21 treatment showed a trend to decrease HMGB1 expression but did not reach to significance. D Quantitative analysis of Western blots showed that IL-10 expression decreased significantly at 24 h in saline-treated TBI mice compared to shams. C21 treatment significantly increased IL-10 expression. E Quantitative analysis showed a significant decrease in eNOS activity as determined by p-eNOS/eNOS ratio after TBI. eNOS activation was significantly increased with C21 treatment compared to saline TBI only. F Quantitative analysis of IL-1β expression demonstrated the corresponding differences in pericontusional cortex at 24 h after TBI. C21 treatment reduced level of IL-1β. G Quantitative analysis of Western blots showed significant increase in TNF-α expression at 24 h after TBI compared to shams. C21 treatment decreased TNF-α level. H Quantitative analysis of cleaved caspse-3 as well as I cleaved PARP immunoblots illustrates significant activation at 24 h after TBI. C21 treatment attenuated cleavage of both pro-apoptotic markers. Values are expressed as mean ± SD (n = 5). *p < 0.05 vs corresponding Sham, **p < 0.01 vs corresponding Sham, ^#p < 0.05 vs corresponding TBI plus C21 treatment, ^##p < 0.01 vs corresponding TBI plus C21 treatment

Fig. 2

Effect of C21 on TUNEL staining in the pericontusional area after TBI. A Representative staining images of TUNEL-positive (green) staining in the pericontusional area of the brain after TBI. B Quantitative TUNEL staining analysis showed that apoptotic cells increased significantly in saline-treated TBI animals compared with the sham group and C21 treatment significantly reduced the apoptotic cells. Magnification × 20, scale bar: 100 μm. Values are expressed as mean ± SD (n = 5). **p < 0.01 vs corresponding Sham, ^##p < 0.01 vs corresponding TBI plus C21 treatment

Fig. 3

Schematic representation of proposed mechanism by which AT2R stimulation with C21 provides neuroprotection in mice after traumatic brain injury. TBI-induced brain degeneration is mediated by down regulation of IL-10 and activation of inflammatory/apoptotic mediators (represented by block arrows). C21 prevents the down regulation of IL-10 and modulates expression of inflammatory/apoptotic mediators (represented by dotted lines). CCI, controlled cortical impact; TBI, traumatic brain injury; RAS, renin–angiotensin system; C21, Compound 21; AT2R, angiotensin II receptor 2; IL-10, interleukin-10