HMGB1-triggered inflammation inhibition of notoginseng leaf triterpenes against cerebral ischemia and reperfusion injury via MAPK and NF-κB signaling pathways

Abstract

Ischemic stroke is a clinically common cerebrovascular disease whose main risks include necrosis, apoptosis and cerebral infarction, all caused by cerebral ischemia and reperfusion (I/R) injury. This process has particular significance for the treatment of stroke patients. Notoginseng leaf triterpenes (PNGL), as a valuable medicine, have been discovered to have neuroprotective effects. However, it was not confirmed that whether PNGL may possess neuroprotective effects against cerebral I/R injury. To explore the neuroprotective effects of PNGL and their underlying mechanisms, a middle cerebral artery occlusion/reperfusion (MCAO/R) model was established. In vivo results suggested that in MCAO/R model rats, PNGL pretreatment (73.0, 146, 292 mg/kg) remarkably decreased infarct volume, reduced brain water content, and improved neurological functions; moreover, PNGL (73.0, 146, 292 mg/kg) significantly alleviated blood-brain barrier (BBB) disruption and inhibited neuronal apoptosis and neuronal loss caused by cerebral I/R injury, while PNGL with a different concertation (146, 292 mg/kg) significantly reduced the concentrations of IL-6, TNF-α, IL-1 β, and HMGB1 in serums in a dose-dependent way, which indicated that inflammation inhibition could be involved in the neuroprotective effects of PNGL. The immunofluorescence and western blot analysis showed PNGL decreased HMGB1 expression, suppressed the HMGB1-triggered inflammation, and inhibited microglia activation (IBA1) in hippocampus and cortex, thus dose-dependently downregulating inflammatory cytokines including VCAM-1, MMP-9, MMP-2, and ICAM-1 concentrations in ischemic brains. Interestingly, PNGL administration (146 mg/kg) significantly downregulated the levels of p-P44/42, p-JNK1/2 and p-P38 MAPK, and also inhibited expressions of the total NF-κB and phosphorylated NF-κB in ischemic brains, which was the downstream pathway triggered by HMGB1. All of these results indicated that the protective effects of PNGL against cerebral I/R injury could be associated with inhibiting HMGB1-triggered inflammation, suppressing the activation of MAPKs and NF-κB, and thus improved cerebral I/R-induced neuropathological changes. This study may offer insight into discovering new active compounds for the treatment of ischemic stroke.

Keywords: HMGB1; MAPK; NF-κB; cerebral ischemia and reperfusion injury; inflammation; notoginseng leaf triterpenes.

Figure 1

The chromatograms and chemical components of notoginseng leaf triterpenes (PNGL) used in experiments via the high performance liquid chromatography. (A) The chromatograms of PNGL samples. (B) Chemical component structures, mainly including ginsenoside Rb1 (No.1), ginsenoside Rb2 (No.3), ginsenoside Rb3 (No.4), and ginsenoside Rc (No.2).

Figure 2

Cerebral blood flow assessed by the laser Doppler blood flow assessment in rats with a middle cerebral artery occlusion/reperfusion MCAO/R injury. (A) The monitored place (core cortex, 2 mm posterior and 6 mm lateral to the bregma). (B,C) Flux mean value of cerebral blood flows in sham and MCAO/R group. (D) The images of cerebral blood flow in the sham group (left) and the model group (right). (E) Flux mean value at different time points. Mean values ± SEM; * p < 0.05, sham group versus MCAO/R group.

Figure 3

Effects of PNGL on infarct volume, brain water content, and neurological deficit scores in MCAO/R injury rats. PNGL improves neurological functions and attenuate brain swellings and infarcts in MCAO/R rats. (A) Representative images of 2, 3, 5-triphenyltetrazolium chloride -stained brain sections from the sham-operated or PNGL-treated animals collected 24 h after infarction; red tissue is healthy; white tissue is infarcted (n = 3–6 in each group). (B) Brain water content in ischemia hemispheres of all groups (n = 4 in each group). (C) Quantitative analysis of the infarct volume (n = 3 in each group). (D) Neurological deficit scores in all groups (n = 10 in each group). Mean values ± SD; * p < 0.05, ** p < 0.01 versus MCAO/R group; # p < 0.05, ## p < 0.01, versus sham group; $$p < 0.01, Nor-ischemic versus Ischemic; NS means no significance, Nor-ischemic versus Ischemic.

Figure 4

Effects of PNGL on BBB disruption and inflammatory cytokines in MCAO/R rats. PNGL alleviates BBB disruption and inflammatory cytokines in MCAO/R rats. (A) Representative images of the Evans blue dye -stained brain sections from the sham-operated or PNGL-treated animals collected 24 h after infarction (4 in each group). (B) The Evans blue leakage content of all groups, measured at 632 nm using spectrophotometry (n = 5 in each group). (C)–(F) the IL-6, TNF-α, IL-1 β, and HMGB1 concentrations, determined by ELISA and specific assay kit (n = 7–8 in each group). Mean values ± SD; * p < 0.05, ** p < 0.01 versus MCAO/R group; ## p < 0.01, versus sham group; $$p < 0.01, Nor-ischemic versus Ischemic; NS means no significance, Nor-ischemic versus Ischemic.

Figure 5

PNGL decreases neuronal apoptosis and neurons loss caused by ischemia. PNGL decreases neuronal apoptosis and neurons loss caused by ischemia in rats. (A) Representative images of Giemsa staining performed in hippocampus CA1, CA3 regions, and cortex regions from ischemic brains, measured by an in situ apoptosis detection kit. (B) Representative images of TUNEL assay performed in hippocampus CA1 and CA3 regions, and cortex regions. (C) The relative neuronal density (%, of sham) in the in hippocampus CA1 and CA3 regions, and cortex regions in all groups (n = 4 in each group). (D) The relative neuronal apoptotic rate levels in the in hippocampus CA1 and CA3 regions, and cortex regions in all groups (n = 3 in each group). Mean values ± SD; * p < 0.05, ** p < 0.01 versus MCAO/R group; ## p < 0.01, versus sham group. Scale bar, 200 μm.

Figure 6

PNGL downregulates inflammatory cytokines and inhibits microglia activation in ischemic brains. PNGL treatment effectively deceased the inflammatory cytokine concentrations, inhibited the microglia activation, and reduced the neuronal loss and apoptosis, thus improving cerebral I/R-induced neuropathological changes by inhibiting neurogenic inflammation. (A) Representative images of IBA1-immunopositive microglia (green) with DAPI (blue) staining from hippocampus CA1, dentate gyrus (DG), and cortex regions in rat brains after MCAO/R injury, measured by immunofluorescence. (B)–(E) The MMP-2, MMP-9, ICAM-1, and VCAM-1 concentrations in hippocampus and cortex in rat brains after MCAO/R injury, determined by ELISA and specific as; (F) the IOD of IBA1-immunopositive microglia (n = 4–6 in each group). Mean values ± SD; * p < 0.05, ** p < 0.01 versus MCAO/R group; # p < 0.05, ## p < 0.01, versus sham group. Scale bar, 100 μm.

Figure 7

Effects of PNGL on the expression levels of phosphorylated NF-κB. PNGL regulated downstream Caspase 3/8/9 signaling pathway in the ischemic brain. PNGL inhibited the NF-κB activation, it regulated neuronal apoptosis and neuroinflammation reactions caused by CIRI. (A) The protein bands of the NF-κB regulated downstream Caspase 3/8/9 signaling pathways in the ischemic brain, which were examined by western blot analysis. (C) the relative NF-κB expression level; (D) the relative expression levels of the phosphorylated NF-κB; (E) (F) (B) The relative expression levels of its downstream Caspase 3/8/9, respectively, quantified and analyzed by using Gel-Pro analyzer software. Mean values ± SD (n = 3); Mean values ± SEM; * p < 0.05, ** p < 0.01 versus MCAO/R group; # p < 0.05, ## p < 0.01, versus sham group.

Figure 8

Effects of PNGL on the expression levels of phosphorylated P44/42, JNK1/2, and P38 of MAPKs signaling pathway in the ischemic brain. PNGL downregulated the expression levels of phosphorylated P44/42, JNK1/2, and P38 of MAPKs. (A) The protein bands of phospho-ERK, phospho-P38, and phospho-JNK, respectively, in the ischemic brain sections were examined by western blot analysis. (B)–(D) The relative expression levels of phosphorylated P44/42, JNK1/2, and P38, respectively, were quantified and analyzed by using Gel-Pro analyzer software. Mean values ± SEM (n = 3); p < 0.05, ** p < 0.01, versus MCAO/R group; # p < 0.05, ## p <0.01 versus sham-operated group.

Figure 9

Effects of PNGL on HMGB1 expression and its HMGB1-triggered inflammation in ischemic brains. PNGL treatment effectively deceased the HMGB1 expression and its HMGB1-triggered inflammation, thus improving cerebral I/R-induced neuropathological changes by inhibiting neurogenic inflammation. (A) Representative images of HMGB1-immunopositive neurons (red) and IBA1-immunopositive microglia (green) with DAPI (blue) staining in hippocampus CA1, dentate gyrus (DG), and cortex regions in rat brains after cerebral I/R, measured by immunofluorescence; scale bar, 100 μm. (B) The protein bands of HMGB1, and the HMGB1-triggered inflammation IL-1β, Cox2 and c-Fos in the ischemic brains were examined by western blot analysis. (C)–(E) The relative expression levels of HMGB1, IL-1β, and Cox2, respectively, quantified and analyzed by using Gel-Pro analyzer software. Mean values ± SEM; * p < 0.05, ** p < 0.01 versus MCAO/R group; # p < 0.05, ## p < 0.01, versus sham group. Scale bar, 100 μm.