Gut commensal Parabacteroides distasonis exerts neuroprotective effects in acute ischemic stroke with hyperuricemia via regulating gut microbiota-gut-brain axis

Abstract

Background: Hyperuricemia is considered as an independent risk factor for acute ischemic stroke (AIS), and some AIS patients are accompanied by an increase in serum uric acid. Recent studies have highlighted the important role of gut microbiota in both hyperuricemia and AIS, but there is little available data on the relationship between gut microbiota and the pathogenesis of AIS with hyperuricemia (HAS).

Methods: Here we profiled the gut microbiota composition in 63 HAS patients and 269 non-HAS patients through 16s rRNA sequencing. Male rat with hyperuricemia were subjected to middle cerebral artery occlusion (MCAO) to establish HAS model and were then treated with Parabacteroides distasonis. Subsequently, the neurological deficit, pathological damages and blood-brain barrier disruption were evaluated. Moreover, the levels of ROS, inflammatory cytokines, NF-𝜿B pathway related protein, and vascular density markers were determined.

Results: There were significant differences of gut microbiota composition between HAS patients and non-HAS patients, and a significant decrease in the abundance of Parabacteroides in HAS patients compared to non-HAS patients. Animal experiments showed that supplementation with P. distasonis increased beneficial commensal bacteria, significantly improved neurological deficits, pathological damages and BBB disruption, as well as reduced the level of serum uric acid in HAS rats. We further demonstrated that P. distasonis treatment decreased ROS level and increased SOD2 level, thereby reducing oxidative stress. Meanwhile, P. distasonis effectively inhibited NF-𝜿B signal pathway and reduced the production of inflammatory cytokines, including TNF-α and IL-1β, alleviating the inflammatory response. Notably, P. distasonis treatment increased the levels of vascular density markers including cluster of differentiation 31 (CD31) and alpha-smooth muscle actin (α-SMA), ameliorating vascular damage in HAS rats.

Conclusions: Together, these findings highlighted the important role of P. distasonis in the pathogenesis of HAS, and its mechanism was involved in the regulation of gut microbiota-gut-brain axis, which implied a novel strategy against HAS.

Keywords: P. distasonis; Gut microbiota; Hyperuricemia associated stroke; Uric acid; Vascular damage.

Fig. 1

Parabacteroides is associated with the prognosis of HAS patients. (A - B) ACE index and Shannon index were used to evaluate alpha diversity of gut microbiota. (C) Venn diagram illustrated the discrepancy and overlap of ASVs in gut microbiota. (D) Stacked bar chart displayed the composition of different taxa and relative abundance at the genus level between two groups. (E) LEfSe analysis showed the taxonomic data with significant differences at the genus level between two groups. LDA scores > 2.0 were shown. (F) Wilcoxon rank-sum test bar plot on genus level showed the significant level of different bacteria between two groups. (G) Comparisons of mRS score between two groups. Data represented as Mean ± SEM. ^*P < 0.05 vs. non-HAS group; ^**P < 0.01 vs. non-HAS group

Fig. 2

P. distasonis reversed abnormal gut microbiota of HAS rats. (A) Comparisons of relative abundance of P. distasonis among Sham group, HAS group and HAS + Pd group. (B) Comparisons of serum UA level among three groups. (C - D) Ace index and Shannon index were used to evaluate α-diversity among three groups. (E) PCoA analysis showed variations of gut microbiota composition among three groups. The significant p value was indicated, and each character represented a sample. (F) Stacked bar chart displayed the composition of different taxa and relative abundance at the genus level among three groups. ^*P < 0.05 vs. Sham group; ^**P < 0.01 vs. Sham group; ^#P < 0.05 vs. HAS group

Fig. 3

P. distasonis improved neurological deficits and ameliorated morphological damages in HAS rats. (A) Comparisons of Zea-Longa scores among Sham group, HAS group and HAS + Pd group. (B) Comparisons of Garcia scores among three groups. (C) Representative images of brain sections stained with TTC among three groups. (D) Percentage of cerebral infarction volume among three groups. (E) Representative images of HE staining, Magnification: 50 ×, Scale bar: 800 μm, and more detailed views of HE staining images, Magnification: 400 ×, Scale bar: 100 μm. Data represented as Mean ± SEM. ^#P < 0.05 vs. HAS group, ^##P < 0.01 vs. HAS group

Fig. 4

P. distasonis alleviated the disruption of the BBB in HAS rats. (A - B) Representative immunofluorescence images of Occludin and ZO-1. Magnification: 400 ×, Scale bar: 100 μm. (C) Representative Western blot images of Occludin and ZO-1. (D - E) Relative quantification of Occludin and ZO-1. The ratios of Occludin/β-actin and ZO-1/β-actin in the Sham group were used as the reference values. Data represented as Mean ± SEM. ^*P < 0.05 vs. Sham group; ^**P < 0.01 vs. Sham group; ^#P < 0.05 vs. HAS group, ^##P < 0.01 vs. HAS group

Fig. 5

P. distasonis mitigated oxidative stress and inflammation in HAS rats. (A) Quantification of relative ROS level. (B) Representative Western blot images of oxidative stress and inflammation related proteins, such as p-p65, p65, IL-1β, TNF-α and SOD2. (C - F) Quantitative analysis of oxidative stress and inflammation related proteins. The ratios of p-p65/p65, IL-1β/β-actin, TNF-α/β-actin and SOD2/β-actin in the Sham group were used as the reference values. Data represented as Mean ± SEM. ^**P < 0.01 vs. Sham group; ^#P < 0.05 vs. HAS group, ^##P < 0.01 vs. HAS group

Fig. 6

P. distasonis ameliorated vascular damage in HAS rats. (A) Representative immunofluorescence images of CD31 and α-SMA. Magnification: 400 ×, Scale bar: 100 μm. (B) Quantification of relative intensity of CD31 positive vessels. (C) Quantification of relative intensity of α-SMA positive vessels. (D) Representative Western blot images of CD31 and α-SMA. (E - F) Relative quantification of CD31 and α-SMA. The ratios of CD31/β-actin and α-SMA/β-actin in the Sham group were used as the reference values. Data represented as Mean ± SEM. ^*P < 0.05 vs. Sham group; ^**P < 0.01 vs. Sham group; ^#P < 0.05 vs. HAS group, ^##P < 0.01 vs. HAS group

Fig. 7

Mechanism diagram. P. distasonis attenuates vascular damage, oxidative stress and inflammation in HAS by regulating gut-brain axis