Effect of HMGB1 and RAGE on brain injury and the protective mechanism of glycyrrhizin in intracranial‑sinus occlusion followed by mechanical thrombectomy recanalization

Abstract

The key to successful treatment of cerebral venous‑sinus occlusion (CVO) is the rapid recanalization of the sinus following venous‑sinus occlusion; however, rapid recanalization of the sinus may also cause secondary cerebral injury. The present study examined mechanical thrombectomy‑related brain injury and the possible molecular mechanisms following CVO recanalization, and investigated the protective effect of glycyrrhizin (GL) in CVO recanalization. The cerebral venous sinus thrombosis (CVST) model was induced in rats using 40% FeCl3. Mechanical thrombectomy was performed at 6 h post‑thrombosis. GL was administered to rats following thromboembolism. Neurological function and brain water content were measured prior to sacrifice of the rats. Serum malondialdehyde, superoxide dismutase and nitric‑oxide synthase concentrations were measured. The expression levels of high‑mobility group box 1 (HMGB1) and receptor of advanced glycation end products (RAGE) and its downstream inflammatory mediators were measured in serum and brain tissues. Rapid CVO recanalization caused brain injury, and the brain parenchymal damage and neurological deficits caused by CVO were not completely restored following recanalization. Similarly, following rapid recanalization in the venous sinus, the expression levels of HMGB1 and RAGE were lower than those in the CVST group, but remained significantly higher than those of the sham group. The combination of mechanical thrombectomy and GL improved cerebral infarction and cerebral edema in rats, and inhibited the extracellular transport of HMGB1, and the expression of downstream inflammatory factors and oxidative‑stress products. The administration of exogenous recombinant HMGB1 reversed the neural protective effects of GL. In conclusion, mechanical thrombectomy subsequent to CVO in rats can cause brain injury following recanalization. HMGB1 and RAGE promote inflammation in the process of brain injury following recanalization. GL has a relatively reliable neuroprotective effect on brain injury by inhibiting HMGB1 and its downstream inflammatory factors, and decreasing oxidative stress.

Figure 1

Process of thrombectomy. (A) A blood-collection needle pierces the superior-sagittal sinus vessel wall at the anterior bone window. (B) Slow insertion of a 0.014-inch micro-guide wire into the venous sinus for mechanical thrombectomy. (C) A gelatin sponge is pressed to stop bleeding following insertion.

Figure 2

Changes in infarct volume, brain water content and ND scores following mechanical thrombectomy, and changes in mRNA and protein expression levels if HMGB1 and RAGE. (A) Quantification of 2,3,5-triphenyltetrazolium chloride staining results, (B) brain water content and (C) neurological function scores, showing that brain parenchymal damage improved following mechanical thrombectomy but was not completely reversed. ^*P<0.05, ^**P<0.01. (D) ELISA results showing that serum concentrations of HMGB1 and RAGE decreased following mechanical thrombectomy. (E) Reverse transcription-quantitative polymerase chain reaction results showing that the mRNA expression levels of HMGB1 and RAGE in paranasal sinus brain tissues were downregulated following mechanical thrombectomy, (F) Western blot analysis and (G) quantitative analysis showing that HMGB1 and RAGE proteins were downregulated in paranasal sinus brain tissues following mechanical thrombectomy. ^*P<0.05 and ^#P<0.05 vs. CVST group. Data are expressed as the mean ± standard deviation. Intergroup differences were analyzed using Student's t-test or one-way analysis of variance. Tukey's post hoc test was used for multiple comparisons among various groups. HMGB1, high-mobility group box 1; RAGE, receptor of advanced glycation end products; CVST, cerebral venous sinus thrombosis; M, mechanical thrombectomy; ND, neurological deficit.

Figure 3

GL protection against venous ischemia/reperfusion injury. Different concentrations of GL were used to interfere with mechanical thrombectomy in rats. (A) 2,3,5-triphenyltetrazolium chloride staining was used to observe infarction and (B) image processing software quantified results. Different concentrations of GL reduced infarct volume. (C) Brain water content in the 4 mg/kg GL group was lower than that in the NS group. (D) Neurological function score in the 4 mg/kg GL group was lower than that in the NS group, and no neurological damage was present in the sham group. Data are expressed as the mean ± standard deviation. Intergroup differences were analyzed using one-way analysis of variance. Tukey's post hoc test was used for multiple comparisons among various groups. ^*P<0.05, ^**P<0.01. GL, glycyrrhizin; NS, normal saline; ND, neurological deficit.

Figure 4

GL inhibits HMGB1 transport and the expression of RAGE following recanalization. (A) Immunofluorescent staining showing differences in the extracellular and intracellular distribution of HMGB1 in paranasal sinus brain tissues. Magnification, ×200. (B) Quantification of HMGB1 fluorescence intensity. (C) Immunofluorescent staining showing changes in the expression of RAGE in paranasal sinus brain tissues. Magnification, ×200. (D) Quantification of RAGE fluorescence intensity. ^***P<0.001. GL, glycyrrhizin; NS, normal saline; HMGB1, high-mobility group box 1; RAGE, receptor of advanced glycation end products.

Figure 5

Expression of HMGB1 and its downstream inflammatory factors following GL inhibition. (A) Changes in serum concentrations of HMGB1 and RAGE at different administration time points. (B) Changes in mRNA expression levels of HMGB1 and RAGE in paranasal sinus brain tissues at different administration time points; (C) Changes in protein expression levels of HMGB1 and RAGE in sinus paraventricular tissues at different administration time points; (D) quantification of protein expression levels. ^*P<0.05 and ^#P<0.05 vs. NS groups. Changes in serum concentrations of (E) TNF-α, IL-1β, IL-6, (F) MDA, (G) SOD and (H) NOS at different doses. ^*P<0.05, ^**P<0.01. Data are expressed as the mean ± standard deviation. Intergroup differences were analyzed using one-way analysis of variance Tukey's post hoc test was used for multiple comparisons among various groups. GL, glycyrrhizin; NS, normal saline; HMGB1, high-mobility group box 1; RAGE, receptor of advanced glycation end products; TNF-α, tumor necrosis factor-α; IL, interleukin; MDA, malondialdehyde; SOD, superoxide dismutase; NOS, nitric oxide synthase.

Figure 6

GL protects against brain damage following CVO recanalization by antagonizing HMGB1. (A) Changes in cerebral infarction volume in rHMGB1-treated or untreated rats. Changes in (B) HMGB1 and RAGE, (C) TNF-α, IL-1β, IL-6, (D) MDA, (E) NOS and (F) SOD in rHMGB1-treated or untreated rats. Data are expressed as the mean ± standard deviation. Intergroup differences were analyzed using one-way analysis of variance. Tukey's post hoc test was used for multiple comparisons among various groups. ^*P<0.05, ^**P<0.01. rHMGB1, recombinant high mobility group box 1; RAGE, receptor for advanced glycation end products; NS, normal saline; GL, glycyrrhizin; TNF-α, tumor necrosis factor-α; IL, interleukin; MDA, malondialdehyde; SOD, superoxide dismutase; NOS, nitric oxide synthase.